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DNA For example, in transcription, when a cell uses the information in a gene, the sequence is copied into a complementary RNA sequence through the attraction between the and the correct RNA nucleotides. Usually, this RNA copy is then used to make a matching protein sequence in a process called translation, which depends on the same interaction between RNA nucleotides. In alternative fashion, a cell may simply copy its genetic information in a process called replication. The details of these functions are covered in other articles; here the focus is on the interactions between and other molecules that mediate the function of the genome. Genomic is tightly and orderly packed in the process called condensation, to fit the small available volumes of the cell. In eukaryotes, is located in the cell nucleus, with small amounts in mitochondria and chloroplasts. In prokaryotes, the is held within an irregularly shaped body in the cytoplasm called the nucleoid. The genetic information in a genome is held within genes, and the complete set of this information in an organism is called its genotype. A gene is a unit of heredity and is a region of that influences a particular characteristic in an organism. Genes contain an open reading frame that can be transcribed, and regulatory sequences such as promoters and enhancers, which control transcription of the open reading frame. In many species, only a small fraction of the total sequence of the genome encodes protein. For example, only about 1 | https://en.wikipedia.org/wiki?curid=7955 |
DNA 5% of the human genome consists of protein-coding exons, with over 50% of human consisting of non-coding repetitive sequences. The reasons for the presence of so much noncoding in eukaryotic genomes and the extraordinary differences in genome size, or "C-value", among species, represent a long-standing puzzle known as the "C-value enigma". However, some sequences that do not code protein may still encode functional non-coding RNA molecules, which are involved in the regulation of gene expression. Some noncoding sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes but are important for the function and stability of chromosomes. An abundant form of noncoding in humans are pseudogenes, which are copies of genes that have been disabled by mutation. These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence. A gene is a sequence of that contains genetic information and can influence the phenotype of an organism. Within a gene, the sequence of bases along a strand defines a messenger RNA sequence, which then defines one or more protein sequences. The relationship between the nucleotide sequences of genes and the amino-acid sequences of proteins is determined by the rules of translation, known collectively as the genetic code | https://en.wikipedia.org/wiki?curid=7955 |
DNA The genetic code consists of three-letter 'words' called "codons" formed from a sequence of three nucleotides (e.g. ACT, CAG, TTT). In transcription, the codons of a gene are copied into messenger RNA by RNA polymerase. This RNA copy is then decoded by a ribosome that reads the RNA sequence by base-pairing the messenger RNA to transfer RNA, which carries amino acids. Since there are 4 bases in 3-letter combinations, there are 64 possible codons (4 combinations). These encode the twenty standard amino acids, giving most amino acids more than one possible codon. There are also three 'stop' or 'nonsense' codons signifying the end of the coding region; these are the TAA, TGA, and TAG codons. Cell division is essential for an organism to grow, but, when a cell divides, it must replicate the in its genome so that the two daughter cells have the same genetic information as their parent. The double-stranded structure of provides a simple mechanism for replication. Here, the two strands are separated and then each strand's complementary sequence is recreated by an enzyme called polymerase. This enzyme makes the complementary strand by finding the correct base through complementary base pairing and bonding it onto the original strand. As polymerases can only extend a strand in a 5′ to 3′ direction, different mechanisms are used to copy the antiparallel strands of the double helix. In this way, the base on the old strand dictates which base appears on the new strand, and the cell ends up with a perfect copy of its DNA | https://en.wikipedia.org/wiki?curid=7955 |
DNA Naked extracellular (eDNA), most of it released by cell death, is nearly ubiquitous in the environment. Its concentration in soil may be as high as 2 μg/L, and its concentration in natural aquatic environments may be as high at 88 μg/L. Various possible functions have been proposed for eDNA: it may be involved in horizontal gene transfer; it may provide nutrients; and it may act as a buffer to recruit or titrate ions or antibiotics. Extracellular acts as a functional extracellular matrix component in the biofilms of several bacterial species. It may act as a recognition factor to regulate the attachment and dispersal of specific cell types in the biofilm; it may contribute to biofilm formation; and it may contribute to the biofilm's physical strength and resistance to biological stress. Cell-free fetal is found in the blood of the mother, and can be sequenced to determine a great deal of information about the developing fetus. ehas seen increased use in the natural sciences as a tool for monitoring the movements and presence of aquatic life, and the technique has been extended to include terrestrial species. All the functions of depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single sequence. Enzymes can also bind to and of these, the polymerases that copy the base sequence in transcription and replication are particularly important. Structural proteins that bind are well-understood examples of non-specific DNA-protein interactions | https://en.wikipedia.org/wiki?curid=7955 |
DNA Within chromosomes, is held in complexes with structural proteins. These proteins organize the into a compact structure called chromatin. In eukaryotes, this structure involves binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome, which contains two complete turns of double-stranded wrapped around its surface. These non-specific interactions are formed through basic residues in the histones, making ionic bonds to the acidic sugar-phosphate backbone of the DNA, and are thus largely independent of the base sequence. Chemical modifications of these basic amino acid residues include methylation, phosphorylation, and acetylation. These chemical changes alter the strength of the interaction between the and the histones, making the more or less accessible to transcription factors and changing the rate of transcription. Other non-specific DNA-binding proteins in chromatin include the high-mobility group proteins, which bind to bent or distorted DNA. These proteins are important in bending arrays of nucleosomes and arranging them into the larger structures that make up chromosomes. A distinct group of DNA-binding proteins is the DNA-binding proteins that specifically bind single-stranded DNA. In humans, replication protein A is the best-understood member of this family and is used in processes where the double helix is separated, including replication, recombination, and repair | https://en.wikipedia.org/wiki?curid=7955 |
DNA These binding proteins seem to stabilize single-stranded and protect it from forming stem-loops or being degraded by nucleases. In contrast, other proteins have evolved to bind to particular sequences. The most intensively studied of these are the various transcription factors, which are proteins that regulate transcription. Each transcription factor binds to one particular set of sequences and activates or inhibits the transcription of genes that have these sequences close to their promoters. The transcription factors do this in two ways. Firstly, they can bind the RNA polymerase responsible for transcription, either directly or through other mediator proteins; this locates the polymerase at the promoter and allows it to begin transcription. Alternatively, transcription factors can bind enzymes that modify the histones at the promoter. This changes the accessibility of the template to the polymerase. As these targets can occur throughout an organism's genome, changes in the activity of one type of transcription factor can affect thousands of genes. Consequently, these proteins are often the targets of the signal transduction processes that control responses to environmental changes or cellular differentiation and development. The specificity of these transcription factors' interactions with come from the proteins making multiple contacts to the edges of the bases, allowing them to "read" the sequence. Most of these base-interactions are made in the major groove, where the bases are most accessible | https://en.wikipedia.org/wiki?curid=7955 |
DNA Nucleases are enzymes that cut strands by catalyzing the hydrolysis of the phosphodiester bonds. Nucleases that hydrolyse nucleotides from the ends of strands are called exonucleases, while endonucleases cut within strands. The most frequently used nucleases in molecular biology are the restriction endonucleases, which cut at specific sequences. For instance, the EcoRV enzyme shown to the left recognizes the 6-base sequence 5′-GATATC-3′ and makes a cut at the horizontal line. In nature, these enzymes protect bacteria against phage infection by digesting the phage when it enters the bacterial cell, acting as part of the restriction modification system. In technology, these sequence-specific nucleases are used in molecular cloning and fingerprinting. Enzymes called ligases can rejoin cut or broken strands. Ligases are particularly important in lagging strand replication, as they join together the short segments of produced at the replication fork into a complete copy of the template. They are also used in repair and genetic recombination. Topoisomerases are enzymes with both nuclease and ligase activity. These proteins change the amount of supercoiling in DNA. Some of these enzymes work by cutting the helix and allowing one section to rotate, thereby reducing its level of supercoiling; the enzyme then seals the break. Other types of these enzymes are capable of cutting one helix and then passing a second strand of through this break, before rejoining the helix | https://en.wikipedia.org/wiki?curid=7955 |
DNA Topoisomerases are required for many processes involving DNA, such as replication and transcription. Helicases are proteins that are a type of molecular motor. They use the chemical energy in nucleoside triphosphates, predominantly adenosine triphosphate (ATP), to break hydrogen bonds between bases and unwind the double helix into single strands. These enzymes are essential for most processes where enzymes need to access the bases. Polymerases are enzymes that synthesize polynucleotide chains from nucleoside triphosphates. The sequence of their products is created based on existing polynucleotide chains—which are called "templates". These enzymes function by repeatedly adding a nucleotide to the 3′ hydroxyl group at the end of the growing polynucleotide chain. As a consequence, all polymerases work in a 5′ to 3′ direction. In the active site of these enzymes, the incoming nucleoside triphosphate base-pairs to the template: this allows polymerases to accurately synthesize the complementary strand of their template. Polymerases are classified according to the type of template that they use. In replication, DNA-dependent polymerases make copies of polynucleotide chains. To preserve biological information, it is essential that the sequence of bases in each copy are precisely complementary to the sequence of bases in the template strand. Many polymerases have a proofreading activity | https://en.wikipedia.org/wiki?curid=7955 |
DNA Here, the polymerase recognizes the occasional mistakes in the synthesis reaction by the lack of base pairing between the mismatched nucleotides. If a mismatch is detected, a 3′ to 5′ exonuclease activity is activated and the incorrect base removed. In most organisms, polymerases function in a large complex called the replisome that contains multiple accessory subunits, such as the clamp or helicases. RNA-dependent polymerases are a specialized class of polymerases that copy the sequence of an RNA strand into DNA. They include reverse transcriptase, which is a viral enzyme involved in the infection of cells by retroviruses, and telomerase, which is required for the replication of telomeres. For example, HIV reverse transcriptase is an enzyme for AIDS virus replication. Telomerase is an unusual polymerase because it contains its own RNA template as part of its structure. It synthesizes telomeres at the ends of chromosomes. Telomeres prevent fusion of the ends of neighboring chromosomes and protect chromosome ends from damage. Transcription is carried out by a DNA-dependent RNA polymerase that copies the sequence of a strand into RNA. To begin transcribing a gene, the RNA polymerase binds to a sequence of called a promoter and separates the strands. It then copies the gene sequence into a messenger RNA transcript until it reaches a region of called the terminator, where it halts and detaches from the DNA | https://en.wikipedia.org/wiki?curid=7955 |
DNA As with human DNA-dependent polymerases, RNA polymerase II, the enzyme that transcribes most of the genes in the human genome, operates as part of a large protein complex with multiple regulatory and accessory subunits. A helix usually does not interact with other segments of DNA, and in human cells, the different chromosomes even occupy separate areas in the nucleus called "chromosome territories". This physical separation of different chromosomes is important for the ability of to function as a stable repository for information, as one of the few times chromosomes interact is in chromosomal crossover which occurs during sexual reproduction, when genetic recombination occurs. Chromosomal crossover is when two helices break, swap a section and then rejoin. Recombination allows chromosomes to exchange genetic information and produces new combinations of genes, which increases the efficiency of natural selection and can be important in the rapid evolution of new proteins. Genetic recombination can also be involved in repair, particularly in the cell's response to double-strand breaks. The most common form of chromosomal crossover is homologous recombination, where the two chromosomes involved share very similar sequences. Non-homologous recombination can be damaging to cells, as it can produce chromosomal translocations and genetic abnormalities. The recombination reaction is catalyzed by enzymes known as recombinases, such as RAD51 | https://en.wikipedia.org/wiki?curid=7955 |
DNA The first step in recombination is a double-stranded break caused by either an endonuclease or damage to the DNA. A series of steps catalyzed in part by the recombinase then leads to joining of the two helices by at least one Holliday junction, in which a segment of a single strand in each helix is annealed to the complementary strand in the other helix. The Holliday junction is a tetrahedral junction structure that can be moved along the pair of chromosomes, swapping one strand for another. The recombination reaction is then halted by cleavage of the junction and re-ligation of the released DNA. Only strands of like polarity exchange during recombination. There are two types of cleavage: east-west cleavage and north–south cleavage. The north–south cleavage nicks both strands of DNA, while the east–west cleavage has one strand of intact. The formation of a Holliday junction during recombination makes it possible for genetic diversity, genes to exchange on chromosomes, and expression of wild-type viral genomes. contains the genetic information that allows all forms of life to function, grow and reproduce. However, it is unclear how long in the 4-billion-year history of life has performed this function, as it has been proposed that the earliest forms of life may have used RNA as their genetic material. RNA may have acted as the central part of early cell metabolism as it can both transmit genetic information and carry out catalysis as part of ribozymes | https://en.wikipedia.org/wiki?curid=7955 |
DNA This ancient RNA world where nucleic acid would have been used for both catalysis and genetics may have influenced the evolution of the current genetic code based on four nucleotide bases. This would occur, since the number of different bases in such an organism is a trade-off between a small number of bases increasing replication accuracy and a large number of bases increasing the catalytic efficiency of ribozymes. However, there is no direct evidence of ancient genetic systems, as recovery of from most fossils is impossible because survives in the environment for less than one million years, and slowly degrades into short fragments in solution. Claims for older have been made, most notably a report of the isolation of a viable bacterium from a salt crystal 250 million years old, but these claims are controversial. Building blocks of (adenine, guanine, and related organic molecules) may have been formed extraterrestrially in outer space. Complex and RNA organic compounds of life, including uracil, cytosine, and thymine, have also been formed in the laboratory under conditions mimicking those found in outer space, using starting chemicals, such as pyrimidine, found in meteorites. Pyrimidine, like polycyclic aromatic hydrocarbons (PAHs), the most carbon-rich chemical found in the universe, may have been formed in red giants or in interstellar cosmic dust and gas clouds | https://en.wikipedia.org/wiki?curid=7955 |
DNA Methods have been developed to purify from organisms, such as phenol-chloroform extraction, and to manipulate it in the laboratory, such as restriction digests and the polymerase chain reaction. Modern biology and biochemistry make intensive use of these techniques in recombinant technology. Recombinant is a man-made sequence that has been assembled from other sequences. They can be transformed into organisms in the form of plasmids or in the appropriate format, by using a viral vector. The genetically modified organisms produced can be used to produce products such as recombinant proteins, used in medical research, or be grown in agriculture. Forensic scientists can use in blood, semen, skin, saliva or hair found at a crime scene to identify a matching of an individual, such as a perpetrator. This process is formally termed profiling, also called "fingerprinting". In profiling, the lengths of variable sections of repetitive DNA, such as short tandem repeats and minisatellites, are compared between people. This method is usually an extremely reliable technique for identifying a matching DNA. However, identification can be complicated if the scene is contaminated with from several people. profiling was developed in 1984 by British geneticist Sir Alec Jeffreys, and first used in forensic science to convict Colin Pitchfork in the 1988 Enderby murders case | https://en.wikipedia.org/wiki?curid=7955 |
DNA The development of forensic science and the ability to now obtain genetic matching on minute samples of blood, skin, saliva, or hair has led to re-examining many cases. Evidence can now be uncovered that was scientifically impossible at the time of the original examination. Combined with the removal of the double jeopardy law in some places, this can allow cases to be reopened where prior trials have failed to produce sufficient evidence to convince a jury. People charged with serious crimes may be required to provide a sample of for matching purposes. The most obvious defense to matches obtained forensically is to claim that cross-contamination of evidence has occurred. This has resulted in meticulous strict handling procedures with new cases of serious crime. profiling is also used successfully to positively identify victims of mass casualty incidents, bodies or body parts in serious accidents, and individual victims in mass war graves, via matching to family members. profiling is also used in paternity testing to determine if someone is the biological parent or grandparent of a child with the probability of parentage is typically 99.99% when the alleged parent is biologically related to the child. Normal sequencing methods happen after birth, but there are new methods to test paternity while a mother is still pregnant. Deoxyribozymes, also called DNAzymes or catalytic DNA, were first discovered in 1994 | https://en.wikipedia.org/wiki?curid=7955 |
DNA They are mostly single stranded sequences isolated from a large pool of random sequences through a combinatorial approach called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). DNAzymes catalyze variety of chemical reactions including RNA-cleavage, RNA-ligation, amino acids phosphorylation-dephosphorylation, carbon-carbon bond formation, and etc. DNAzymes can enhance catalytic rate of chemical reactions up to 100,000,000,000-fold over the uncatalyzed reaction. The most extensively studied class of DNAzymes is RNA-cleaving types which have been used to detect different metal ions and designing therapeutic agents. Several metal-specific DNAzymes have been reported including the GR-5 DNAzyme (lead-specific), the CA1-3 DNAzymes (copper-specific), the 39E DNAzyme (uranyl-specific) and the NaA43 DNAzyme (sodium-specific). The NaA43 DNAzyme, which is reported to be more than 10,000-fold selective for sodium over other metal ions, was used to make a real-time sodium sensor in cells. Bioinformatics involves the development of techniques to store, data mine, search and manipulate biological data, including nucleic acid sequence data. These have led to widely applied advances in computer science, especially string searching algorithms, machine learning, and database theory. String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides | https://en.wikipedia.org/wiki?curid=7955 |
DNA The sequence may be aligned with other sequences to identify homologous sequences and locate the specific mutations that make them distinct. These techniques, especially multiple sequence alignment, are used in studying phylogenetic relationships and protein function. Data sets representing entire genomes' worth of sequences, such as those produced by the Human Genome Project, are difficult to use without the annotations that identify the locations of genes and regulatory elements on each chromosome. Regions of sequence that have the characteristic patterns associated with protein- or RNA-coding genes can be identified by gene finding algorithms, which allow researchers to predict the presence of particular gene products and their possible functions in an organism even before they have been isolated experimentally. Entire genomes may also be compared, which can shed light on the evolutionary history of particular organism and permit the examination of complex evolutionary events. nanotechnology uses the unique molecular recognition properties of and other nucleic acids to create self-assembling branched complexes with useful properties. is thus used as a structural material rather than as a carrier of biological information. This has led to the creation of two-dimensional periodic lattices (both tile-based and using the "origami" method) and three-dimensional structures in the shapes of polyhedra | https://en.wikipedia.org/wiki?curid=7955 |
DNA Nanomechanical devices and algorithmic self-assembly have also been demonstrated, and these structures have been used to template the arrangement of other molecules such as gold nanoparticles and streptavidin proteins. Because collects mutations over time, which are then inherited, it contains historical information, and, by comparing sequences, geneticists can infer the evolutionary history of organisms, their phylogeny. This field of phylogenetics is a powerful tool in evolutionary biology. If sequences within a species are compared, population geneticists can learn the history of particular populations. This can be used in studies ranging from ecological genetics to anthropology. as a storage device for information has enormous potential since it has much higher storage density compared to electronic devices. However high costs, extremely slow read and write times (memory latency), and insufficient reliability has prevented its practical use. was first isolated by the Swiss physician Friedrich Miescher who, in 1869, discovered a microscopic substance in the pus of discarded surgical bandages. As it resided in the nuclei of cells, he called it "nuclein". In 1878, Albrecht Kossel isolated the non-protein component of "nuclein", nucleic acid, and later isolated its five primary nucleobases. In 1909, Phoebus Levene identified the base, sugar, and phosphate nucleotide unit of the RNA (then named "yeast nucleic acid"). In 1929, Levene identified deoxyribose sugar in "thymus nucleic acid" (DNA) | https://en.wikipedia.org/wiki?curid=7955 |
DNA Levene suggested that consisted of a string of four nucleotide units linked together through the phosphate groups ("tetranucleotide hypothesis"). Levene thought the chain was short and the bases repeated in a fixed order. In 1927, Nikolai Koltsov proposed that inherited traits would be inherited via a "giant hereditary molecule" made up of "two mirror strands that would replicate in a semi-conservative fashion using each strand as a template". In 1928, Frederick Griffith in his experiment discovered that traits of the "smooth" form of "Pneumococcus" could be transferred to the "rough" form of the same bacteria by mixing killed "smooth" bacteria with the live "rough" form. This system provided the first clear suggestion that carries genetic information. In 1933, while studying virgin sea urchin eggs, Jean Brachet suggested that is found in the cell nucleus and that RNA is present exclusively in the cytoplasm. At the time, "yeast nucleic acid" (RNA) was thought to occur only in plants, while "thymus nucleic acid" (DNA) only in animals. The latter was thought to be a tetramer, with the function of buffering cellular pH. In 1937, William Astbury produced the first X-ray diffraction patterns that showed that had a regular structure. In 1943, Oswald Avery, along with co-workers Colin MacLeod and Maclyn McCarty, identified as the transforming principle, supporting Griffith's suggestion (Avery–MacLeod–McCarty experiment) | https://en.wikipedia.org/wiki?curid=7955 |
DNA DNA's role in heredity was confirmed in 1952 when Alfred Hershey and Martha Chase in the Hershey–Chase experiment showed that is the genetic material of the enterobacteria phage T2. Late in 1951, Francis Crick started working with James Watson at the Cavendish Laboratory within the University of Cambridge. In February 1953, Linus Pauling and Robert Corey proposed a model for nucleic acids containing three intertwined chains, with the phosphates near the axis, and the bases on the outside. In May 1952, Raymond Gosling a graduate student working under the supervision of Rosalind Franklin took an X-ray diffraction image, labeled as "Photo 51", at high hydration levels of DNA. This photo was given to Watson and Crick by Maurice Wilkins and was critical to their obtaining the correct structure of DNA. Franklin told Crick and Watson that the backbones had to be on the outside. Before then, Linus Pauling, and Watson and Crick, had erroneous models with the chains inside and the bases pointing outwards. Her identification of the space group for crystals revealed to Crick that the two strands were antiparallel. In February 1953, Watson and Crick completed their model, which is now accepted as the first correct model of the double-helix of DNA. On 28 February 1953 Crick interrupted patrons' lunchtime at The Eagle pub in Cambridge to announce that he and Watson had "discovered the secret of life" | https://en.wikipedia.org/wiki?curid=7955 |
DNA In the 25 April 1953 issue of the journal "Nature", were published a series of five articles giving the Watson and Crick double-helix structure DNA, and evidence supporting it. The structure was reported in a letter titled ""MOLECULAR STRUCTURE OF NUCLEIC ACIDS A Structure for Deoxyribose Nucleic Acid"", in which they said, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Followed by a letter from Franklin and Gosling, which was the first publication of their own X-ray diffraction data, and of their original analysis method. Then followed a letter by Wilkins, and two of his colleagues, which contained an analysis of "in vivo" B-X-ray patterns, and supported the presence "in vivo" of the Watson and Crick structure. In 1962, after Franklin's death, Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine. Nobel Prizes are awarded only to living recipients. A debate continues about who should receive credit for the discovery. In an influential presentation in 1957, Crick laid out the central dogma of molecular biology, which foretold the relationship between DNA, RNA, and proteins, and articulated the "adaptor hypothesis". Final confirmation of the replication mechanism that was implied by the double-helical structure followed in 1958 through the Meselson–Stahl experiment | https://en.wikipedia.org/wiki?curid=7955 |
DNA Further work by Crick and co-workers showed that the genetic code was based on non-overlapping triplets of bases, called codons, allowing Har Gobind Khorana, Robert W. Holley, and Marshall Warren Nirenberg to decipher the genetic code. These findings represent the birth of molecular biology. | https://en.wikipedia.org/wiki?curid=7955 |
DNA ligase is a specific type of enzyme, a ligase, () that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms (such as IV) may specifically repair double-strand breaks (i.e. a break in both complementary strands of DNA). Single-strand breaks are repaired by using the complementary strand of the double helix as a template, with creating the final phosphodiester bond to fully repair the DNA. is used in both DNA repair and DNA replication (see "Mammalian ligases"). In addition, has extensive use in molecular biology laboratories for recombinant DNA experiments (see "Research applications"). Purified is used in gene cloning to join DNA molecules together to form recombinant DNA. The mechanism of is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide ("acceptor"), with the 5' phosphate end of another ("donor"). Two ATP molecules are consumed for each phosphodiester bond formed. AMP is required for the ligase reaction, which proceeds in four steps: Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required. The "E. coli" is encoded by the "lig" gene. in "E. coli", as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently. The activity of E. coli can be enhanced by DNA polymerase at the right concentrations. Enhancement only works when the concentrations of the DNA polymerase 1 are much lower than the DNA fragments to be ligated. When the concentrations of Pol I DNA polymerases are higher, it has an adverse effect on E. coli The from bacteriophage T4 (a bacteriophage that infects "Escherichia coli" bacteria). The T4 ligase is the most-commonly used in laboratory research. It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. It can also ligate blunt-ended DNA with much greater efficiency than "E. coli" DNA ligase. Unlike "E. coli" DNA ligase, T4 cannot utilize NAD and it has an absolute requirement for ATP as a cofactor. Some engineering has been done to improve the "in vitro" activity of T4 DNA ligase; one successful approach, for example, tested T4 fused to several alternative DNA binding proteins and found that the constructs with either p50 or NF-kB as fusion partners were over 160% more active in blunt-end ligations for cloning purposes than wild type T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vector would use about 0.01 (sticky ends) to 1 (blunt ends) units of ligase. The optimal incubation temperature for T4 is 16 °C | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase In mammals, there are four specific types of ligase. from eukaryotes and some microbes uses adenosine triphosphate (ATP) rather than NAD. Derived from a thermophilic bacterium, the enzyme is stable and active at much higher temperatures than conventional DNA ligases. Its half-life is 48 hours at 65 °C and greater than 1 hour at 95 °C. Ampligase DNA Ligase has been shown to be active for at least 500 thermal cycles (94 °C/80 °C) or 16 hours of cycling. This exceptional thermostability permits extremely high hybridization stringency and ligation specificity. There are at least three different units used to measure the activity of DNA ligase: DNA ligases have become indispensable tools in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids. Controlling the optimal temperature is a vital aspect of performing efficient recombination experiments involving the ligation of cohesive-ended fragments. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4), which is most active at 37 °C. However, for optimal ligation efficiency with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature T of the sticky ends being ligated, the homologous pairing of the sticky ends will not be stable because the high temperature disrupts hydrogen bonding | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase A ligation reaction is most efficient when the sticky ends are already stably annealed, and disruption of the annealing ends would therefore result in low ligation efficiency. The shorter the overhang, the lower the T. Since blunt-ended DNA fragments have no cohesive ends to anneal, the melting temperature is not a factor to consider within the normal temperature range of the ligation reaction. The limiting factor in blunt end ligation is not the activity of the ligase but rather the number of alignments between DNA fragment ends that occur. The most efficient ligation temperature for blunt-ended DNA would therefore be the temperature at which the greatest number of alignments can occur. The majority of blunt-ended ligations are carried out at 14-25 °C overnight. The absence of stably annealed ends also means that the ligation efficiency is lowered, requiring a higher ligase concentration to be used. A novel use of can be seen in the field of nano chemistry, specifically in DNA origami. DNA based self-assembly principles have proven useful for organizing nanoscale objects, such as biomolecules, nanomachines, nanoelectronic and photonic component. Assembly of such nano structure requires the creation of an intricate mesh of DNA molecules. Although DNA self-assembly is possible without any outside help using different substrates such as provision of catatonic surface of Aluminium foil, can provide the enzymatic assistance that is required to make DNA lattice structure from DNA over hangs | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase The first was purified and characterized in 1967 by the Gellert, Lehman, Richardson, and Hurwitz laboratories. It was first purified and characterized by Weiss and Richardson using a six-step chromatographic-fractionation process beginning with elimination of cell debris and addition of streptomycin, followed by several Diethylaminoethyl (DEAE)-cellulose column washes and a final phosphocellulose fractionation. The final extract contained 10% of the activity initially recorded in the "E. coli "media; along the process it was discovered that ATP and Mg++ were necessary to optimize the reaction. The common commercially available DNA ligases were originally discovered in bacteriophage T4, "E. coli" and other bacteria. Genetic deficiencies in human DNA ligases have been associated with clinical syndromes marked by immunodeficiency, radiation sensitivity, and developmental abnormalities, LIG4 syndrome (Ligase IV syndrome) is a rare disease associated with mutations in 4 and interferes with dsDNA break-repair mechanisms. Ligase IV syndrome causes immunodeficiency in individuals and is commonly associated with microcephaly and marrow hypoplasia. A list of prevalent diseases caused by lack of or malfunctioning of is as follows. Xeroderma pigmentosum, which is commonly known as XP, is an inherited condition characterized by an extreme sensitivity to ultraviolet (UV) rays from sunlight. This condition mostly affects the eyes and areas of skin exposed to the sun | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase Some affected individuals also have problems involving the nervous system. Mutations in the ATM gene cause ataxia–telangiectasia. The ATM gene provides instructions for making a protein that helps control cell division and is involved in DNA repair. This protein plays an important role in the normal development and activity of several body systems, including the nervous system and immune system. The ATM protein assists cells in recognizing damaged or broken DNA strands and coordinates DNA repair by activating enzymes that fix the broken strands. Efficient repair of damaged DNA strands helps maintain the stability of the cell's genetic information. Affected children typically develop difficulty walking, problems with balance and hand coordination, involuntary jerking movements (chorea), muscle twitches (myoclonus), and disturbances in nerve function (neuropathy). The movement problems typically cause people to require wheelchair assistance by adolescence. People with this disorder also have slurred speech and trouble moving their eyes to look side-to-side (oculomotor apraxia). Fanconi anemia (FA) is a rare, inherited blood disorder that leads to bone marrow failure. FA prevents bone marrow from making enough new blood cells for the body to work normally. FA also can cause the bone marrow to make many faulty blood cells. This can lead to serious health problems, such as leukemia | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase Bloom syndrome results in skin that is sensitive to sun exposure, and usually the development of a butterfly-shaped patch of reddened skin across the nose and cheeks. A skin rash can also appear on other areas that are typically exposed to the sun, such as the back of the hands and the forearms. Small clusters of enlarged blood vessels (telangiectases) often appear in the rash; telangiectases can also occur in the eyes. Other skin features include patches of skin that are lighter or darker than the surrounding areas (hypopigmentation or hyperpigmentation respectively). These patches appear on areas of the skin that are not exposed to the sun, and their development is not related to the rashes. In recent studies, human I was used in Computer-aided drug design to identify inhibitors as possible therapeutic agents to treat cancer. Since excessive cell growth is a hallmark of cancer development, targetes chemotherapy that disrupts the functioning of can impede adjuvant cancer forms. Furthermore, it has been shown that can be broadly divided into two categories namely, ATP dependent and NAD+ dependent. Previous research has shown that although NAD-dependent DNA ligases have been discovered in sporadic cellular or viral niches outside the bacterial domain of life, there is no instance in which a NAD-dependent ligase is present in a eukaryal organism | https://en.wikipedia.org/wiki?curid=8697 |
DNA ligase The narrow phylogenetic distribution, unique substrate specificity, and distinctive domain structure of NAD+ depandant compared with ATP-dependent human DNA ligases recommend the NAD ligases as targets for the development of new antibacterial drugs. | https://en.wikipedia.org/wiki?curid=8697 |
DARPA The Defense Advanced Research Projects Agency (DARPA) is an agency of the United States Department of Defense responsible for the development of emerging technologies for use by the military. Originally known as the Advanced Research Projects Agency (ARPA), the agency was created in February 1958 by President Dwight D. Eisenhower in response to the Soviet launching of Sputnik 1 in 1957. By collaborating with academic, industry, and government partners, formulates and executes research and development projects to expand the frontiers of technology and science, often beyond immediate U.S. military requirements. DARPA-funded projects have provided significant technologies that influenced many non-military fields, such as computer networking and the basis for the modern Internet, and graphical user interfaces in information technology. is independent of other military research and development and reports directly to senior Department of Defense management. comprises approximately 220 government employees in six technical offices, including nearly 100 program managers, who together oversee about 250 research and development programs. The name of the organization first changed from its founding name ARPA to in March 1972, briefly changing back to ARPA in February 1993, only to revert to in March 1996. Currently, their mission statement is "to make pivotal investments in breakthrough technologies for national security". The creation of the Advanced Research Projects Agency (ARPA) was authorized by President Dwight D | https://en.wikipedia.org/wiki?curid=8957 |
DARPA Eisenhower in 1958 for the purpose of forming and executing research and development projects to expand the frontiers of technology and science, and able to reach far beyond immediate military requirements, the two relevant acts being the Supplemental Military Construction Authorization (Air Force) (Public Law 85-325) and Department of Defense Directive 5105.15, in February 1958. Its creation was directly attributed to the launching of Sputnik and to U.S. realization that the Soviet Union had developed the capacity to rapidly exploit military technology. Initial funding of ARPA was $520 million. ARPA's first director, Roy Johnson, left a $160,000 management job at General Electric for an $18,000 job at ARPA. Herbert York from Lawrence Livermore National Laboratory was hired as his scientific assistant. Johnson and York were both keen on space projects, but when NASA was established later in 1958 all space projects and most of ARPA's funding were transferred to it. Johnson resigned and ARPA was repurposed to do "high-risk", "high-gain", "far out" basic research, a posture that was enthusiastically embraced by the nation's scientists and research universities. ARPA's second director was Brigadier General Austin W. Betts, who resigned in early 1961. He was succeeded by Jack Ruina who served until 1963. Ruina, the first scientist to administer ARPA, managed to raise its budget to $250 million. It was Ruina who hired J. C. R | https://en.wikipedia.org/wiki?curid=8957 |
DARPA Licklider as the first administrator of the Information Processing Techniques Office, which played a vital role in creation of ARPANET, the basis for the future Internet. Additionally, the political and defense communities recognized the need for a high-level Department of Defense organization to formulate and execute R&D projects that would expand the frontiers of technology beyond the immediate and specific requirements of the Military Services and their laboratories. In pursuit of this mission, has developed and transferred technology programs encompassing a wide range of scientific disciplines that address the full spectrum of national security needs. From 1958 to 1965, ARPA's emphasis centered on major national issues, including space, ballistic missile defense, and nuclear test detection. During 1960, all of its civilian space programs were transferred to the National Aeronautics and Space Administration (NASA) and the military space programs to the individual services. This allowed ARPA to concentrate its efforts on the Project Defender (defense against ballistic missiles), Project Vela (nuclear test detection), and Project AGILE (counterinsurgency R&D) programs, and to begin work on computer processing, behavioral sciences, and materials sciences. The DEFENDER and AGILE programs formed the foundation of sensor, surveillance, and directed energy R&D, particularly in the study of radar, infrared sensing, and x-ray/gamma ray detection | https://en.wikipedia.org/wiki?curid=8957 |
DARPA ARPA at this point (1959) played an early role in Transit (also called NavSat) a predecessor to the Global Positioning System (GPS). "Fast-forward to 1959 when a joint effort between and the Johns Hopkins Applied Physics Laboratory began to fine-tune the early explorers’ discoveries. TRANSIT, sponsored by the Navy and developed under the leadership of Dr. Richard Kirschner at Johns Hopkins, was the first satellite positioning system." During the late 1960s, with the transfer of these mature programs to the Services, ARPA redefined its role and concentrated on a diverse set of relatively small, essentially exploratory research programs. The agency was renamed the Defense Advanced Research Projects Agency (DARPA) in 1972, and during the early 1970s, it emphasized direct energy programs, information processing, and tactical technologies. Concerning information processing, made great progress, initially through its support of the development of time-sharing (all modern operating systems rely on concepts invented for the Multics system, developed by a cooperation among Bell Labs, General Electric and MIT, which supported by funding Project MAC at MIT with an initial two-million-dollar grant). supported the evolution of the ARPANET (the first wide-area packet switching network), Packet Radio Network, Packet Satellite Network and ultimately, the Internet and research in the artificial intelligence fields of speech recognition and signal processing, including parts of Shakey the robot | https://en.wikipedia.org/wiki?curid=8957 |
DARPA also funded the development of the Douglas Engelbart's NLS computer system and The Mother of All Demos; and the Aspen Movie Map, which was probably the first hypermedia system and an important precursor of virtual reality. The Mansfield Amendment of 1973 expressly limited appropriations for defense research (through ARPA/DARPA) only to projects with direct military application. Some contend that the amendment devastated American science, since ARPA/was a major funding source for basic science projects of the time; the National Science Foundation never made up the difference as expected. The resulting "brain drain" is also credited with boosting the development of the fledgling personal computer industry. Some young computer scientists left the universities to startups and private research laboratories such as Xerox PARC. Between 1976 and 1981, DARPA's major projects were dominated by air, land, sea, and space technology, tactical armor and anti-armor programs, infrared sensing for space-based surveillance, high-energy laser technology for space-based missile defense, antisubmarine warfare, advanced cruise missiles, advanced aircraft, and defense applications of advanced computing. These large-scale technological program demonstrations were joined by integrated circuit research, which resulted in submicrometer electronic technology and electron devices that evolved into the Very-Large-Scale Integration (VLSI) Program and the Congressionally-mandated charged particle beam program | https://en.wikipedia.org/wiki?curid=8957 |
DARPA Many of the successful programs were transitioned to the Services, such as the foundation technologies in automatic target recognition, space based sensing, propulsion, and materials that were transferred to the Strategic Defense Initiative Organization (SDIO), later known as the Ballistic Missile Defense Organization (BMDO), now titled the Missile Defense Agency (MDA). During the 1980s, the attention of the Agency was centered on information processing and aircraft-related programs, including the National Aerospace Plane (NASP) or Hypersonic Research Program. The Strategic Computing Program enabled to exploit advanced processing and networking technologies and to rebuild and strengthen relationships with universities after the Vietnam War. In addition, began to pursue new concepts for small, lightweight satellites (LIGHTSAT) and directed new programs regarding defense manufacturing, submarine technology, and armor/anti-armor. On February 4, 2004 the agency shut down its so called "LifeLog Project". The project's aim would have been, "to gather in a single place just about everything an individual says, sees or does". On October 28, 2009 the agency broke ground on a new facility in Arlington, Virginia a few miles from the Pentagon. In fall 2011, hosted the 100-Year Starship Symposium with the aim of getting the public to start thinking seriously about interstellar travel | https://en.wikipedia.org/wiki?curid=8957 |
DARPA On June 5, 2016, NASA and announced that it planned to build new X-planes with NASA's plan setting to create a whole series of X planes over the next 10 years. Between 2014 and 2016, shepherded the first machine-to-machine computer security competition, the Cyber Grand Challenge (CGC), bringing a group of top-notch computer security experts to search for security vulnerabilities, exploit them, and create fixes that patch those vulnerabilities in a fully-automated fashion. In June 2018, leaders demonstrated a number of new technologies that were developed within the framework of the GXV-T program. The goal of this program is to create a lightly armored combat vehicle of not very large dimensions, which, due to maneuverability and other tricks, can successfully resist modern anti-tank weapon systems. has six technical offices that manage the agency's research portfolio, and two additional support offices that manage special projects and transition efforts. All offices report to the director: A 1991 reorganization created several offices which existed throughout the early 1990s: Reorganization in 2010 merged two offices: A list of DARPA's active and archived projects is available on the agency's website. Because of the agency's fast pace, programs constantly start and stop based on the needs of the U.S. government. Structured information about some of the DARPA's contracts and projects is publicly available. is well known as a high-tech government agency, and as such has many appearances in popular fiction | https://en.wikipedia.org/wiki?curid=8957 |
DARPA Some realistic references to in fiction are as "ARPA" in "Tom Swift and the Visitor from Planet X" (consults on a technical threat), in episodes of television program "The West Wing" (the ARPA-distinction), the television program "Numb3rs" | https://en.wikipedia.org/wiki?curid=8957 |
Genetic engineering Genetic engineering, also called Genetic modification or Genetic manipulation, is the direct manipulation of an organism's genes using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome. An organism that is generated through genetic engineering is considered to be genetically modified (GM) and the resulting entity is a genetically modified organism (GMO). The first GMO was a bacterium generated by Herbert Boyer and Stanley Cohen in 1973. Rudolf Jaenisch created the first GM animal when he inserted foreign DNA into a mouse in 1974. The first company to focus on genetic engineering, Genentech, was founded in 1976 and started the production of human proteins. Genetically engineered human insulin was produced in 1978 and insulin-producing bacteria were commercialised in 1982 | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Genetically modified food has been sold since 1994, with the release of the Flavr Savr tomato. The Flavr Savr was engineered to have a longer shelf life, but most current GM crops are modified to increase resistance to insects and herbicides. GloFish, the first GMO designed as a pet, was sold in the United States in December 2003. In 2016 salmon modified with a growth hormone were sold. has been applied in numerous fields including research, medicine, industrial biotechnology and agriculture. In research GMOs are used to study gene function and expression through loss of function, gain of function, tracking and expression experiments. By knocking out genes responsible for certain conditions it is possible to create animal model organisms of human diseases. As well as producing hormones, vaccines and other drugs genetic engineering has the potential to cure genetic diseases through gene therapy. The same techniques that are used to produce drugs can also have industrial applications such as producing enzymes for laundry detergent, cheeses and other products. The rise of commercialised genetically modified crops has provided economic benefit to farmers in many different countries, but has also been the source of most of the controversy surrounding the technology. This has been present since its early use; the first field trials were destroyed by anti-GM activists | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Although there is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, GM food safety is a leading concern with critics. Gene flow, impact on non-target organisms, control of the food supply and intellectual property rights have also been raised as potential issues. These concerns have led to the development of a regulatory framework, which started in 1975. It has led to an international treaty, the Cartagena Protocol on Biosafety, that was adopted in 2000. Individual countries have developed their own regulatory systems regarding GMOs, with the most marked differences occurring between the US and Europe. is a process that alters the genetic structure of an organism by either removing or introducing DNA. Unlike traditional animal and plant breeding, which involves doing multiple crosses and then selecting for the organism with the desired phenotype, genetic engineering takes the gene directly from one organism and delivers it to the other. This is much faster, can be used to insert any genes from any organism (even ones from different domains) and prevents other undesirable genes from also being added. could potentially fix severe genetic disorders in humans by replacing the defective gene with a functioning one. It is an important tool in research that allows the function of specific genes to be studied. Drugs, vaccines and other products have been harvested from organisms engineered to produce them | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Crops have been developed that aid food security by increasing yield, nutritional value and tolerance to environmental stresses. The DNA can be introduced directly into the host organism or into a cell that is then fused or hybridised with the host. This relies on recombinant nucleic acid techniques to form new combinations of heritable genetic material followed by the incorporation of that material either indirectly through a vector system or directly through micro-injection, macro-injection or micro-encapsulation. does not normally include traditional breeding, in vitro fertilisation, induction of polyploidy, mutagenesis and cell fusion techniques that do not use recombinant nucleic acids or a genetically modified organism in the process. However, some broad definitions of genetic engineering include selective breeding. Cloning and stem cell research, although not considered genetic engineering, are closely related and genetic engineering can be used within them. Synthetic biology is an emerging discipline that takes genetic engineering a step further by introducing artificially synthesised material into an organism. Such synthetic DNA as Artificially Expanded Genetic Information System and Hachimoji DNA is made in this new field. Plants, animals or microorganisms that have been changed through genetic engineering are termed genetically modified organisms or GMOs. If genetic material from another species is added to the host, the resulting organism is called transgenic | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering If genetic material from the same species or a species that can naturally breed with the host is used the resulting organism is called cisgenic. If genetic engineering is used to remove genetic material from the target organism the resulting organism is termed a knockout organism. In Europe genetic modification is synonymous with genetic engineering while within the United States of America and Canada genetic modification can also be used to refer to more conventional breeding methods. Humans have altered the genomes of species for thousands of years through selective breeding, or artificial selection as contrasted with natural selection. More recently, mutation breeding has used exposure to chemicals or radiation to produce a high frequency of random mutations, for selective breeding purposes. as the direct manipulation of DNA by humans outside breeding and mutations has only existed since the 1970s. The term "genetic engineering" was first coined by Jack Williamson in his science fiction novel "Dragon's Island", published in 1951 – one year before DNA's role in heredity was confirmed by Alfred Hershey and Martha Chase, and two years before James Watson and Francis Crick showed that the DNA molecule has a double-helix structure – though the general concept of direct genetic manipulation was explored in rudimentary form in Stanley G. Weinbaum's 1936 science fiction story "Proteus Island" | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering In 1972, Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus. In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an "Escherichia coli" bacterium. A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world's first transgenic animal These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe. In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and Robert Swanson and a year later the company produced a human protein (somatostatin) in "E.coli". Genentech announced the production of genetically engineered human insulin in 1978. In 1980, the U.S. Supreme Court in the "Diamond v. Chakrabarty" case ruled that genetically altered life could be patented. The insulin produced by bacteria was approved for release by the Food and Drug Administration (FDA) in 1982. In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering government authorisation to perform field tests with the ice-minus strain of "Pseudomonas syringae" to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges. In 1987, the ice-minus strain of "P. syringae" became the first genetically modified organism (GMO) to be released into the environment when a strawberry field and a potato field in California were sprayed with it. Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher". The first field trials of genetically engineered plants occurred in France and the US in 1986, tobacco plants were engineered to be resistant to herbicides. The People's Republic of China was the first country to commercialise transgenic plants, introducing a virus-resistant tobacco in 1992. In 1994 Calgene attained approval to commercially release the first genetically modified food, the Flavr Savr, a tomato engineered to have a longer shelf life. In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialised in Europe. In 1995, Bt Potato was approved safe by the Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the US | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering In 2009 11 transgenic crops were grown commercially in 25 countries, the largest of which by area grown were the US, Brazil, Argentina, India, Canada, China, Paraguay and South Africa. In 2010, scientists at the J. Craig Venter Institute created the first synthetic genome and inserted it into an empty bacterial cell. The resulting bacterium, named Mycoplasma laboratorium, could replicate and produce proteins. Four years later this was taken a step further when a bacterium was developed that replicated a plasmid containing a unique base pair, creating the first organism engineered to use an expanded genetic alphabet. In 2012, Jennifer Doudna and Emmanuelle Charpentier collaborated to develop the CRISPR/Cas9 system, a technique which can be used to easily and specifically alter the genome of almost any organism. Creating a GMO is a multi-step process. Genetic engineers must first choose what gene they wish to insert into the organism. This is driven by what the aim is for the resultant organism and is built on earlier research. Genetic screens can be carried out to determine potential genes and further tests then used to identify the best candidates. The development of microarrays, transcriptomics and genome sequencing has made it much easier to find suitable genes. Luck also plays its part; the round-up ready gene was discovered after scientists noticed a bacterium thriving in the presence of the herbicide. The next step is to isolate the candidate gene | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering The cell containing the gene is opened and the DNA is purified. The gene is separated by using restriction enzymes to cut the DNA into fragments or polymerase chain reaction (PCR) to amplify up the gene segment. These segments can then be extracted through gel electrophoresis. If the chosen gene or the donor organism's genome has been well studied it may already be accessible from a genetic library. If the DNA sequence is known, but no copies of the gene are available, it can also be artificially synthesised. Once isolated the gene is ligated into a plasmid that is then inserted into a bacterium. The plasmid is replicated when the bacteria divide, ensuring unlimited copies of the gene are available. Before the gene is inserted into the target organism it must be combined with other genetic elements. These include a promoter and terminator region, which initiate and end transcription. A selectable marker gene is added, which in most cases confers antibiotic resistance, so researchers can easily determine which cells have been successfully transformed. The gene can also be modified at this stage for better expression or effectiveness. These manipulations are carried out using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning. There are a number of techniques used to insert genetic material into the host genome. Some bacteria can naturally take up foreign DNA. This ability can be induced in other bacteria via stress (e.g | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering thermal or electric shock), which increases the cell membrane's permeability to DNA; up-taken DNA can either integrate with the genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cell's nuclear envelope directly into the nucleus, or through the use of viral vectors. In plants the DNA is often inserted using "Agrobacterium"-mediated recombination, taking advantage of the "Agrobacterium"s T-DNA sequence that allows natural insertion of genetic material into plant cells. Other methods include biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells, and electroporation, which involves using an electric shock to make the cell membrane permeable to plasmid DNA. As only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through the use of tissue culture. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary. Selectable markers are used to easily differentiate transformed from untransformed cells. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be expressed at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. The new genetic material can be inserted randomly within the host genome or targeted to a specific location. The technique of gene targeting uses homologous recombination to make desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced through genome editing. Genome editing uses artificially engineered nucleases that create specific double-stranded breaks at desired locations in the genome, and use the cell's endogenous mechanisms to repair the induced break by the natural processes of homologous recombination and nonhomologous end-joining. There are four families of engineered nucleases: meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and the Cas9-guideRNA system (adapted from CRISPR) | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering TALEN and CRISPR are the two most commonly used and each has its own advantages. TALENs have greater target specificity, while CRISPR is easier to design and more efficient. In addition to enhancing gene targeting, engineered nucleases can be used to introduce mutations at endogenous genes that generate a gene knockout. has applications in medicine, research, industry and agriculture and can be used on a wide range of plants, animals and microorganisms. Bacteria, the first organisms to be genetically modified, can have plasmid DNA inserted containing new genes that code for medicines or enzymes that process food and other substrates. Plants have been modified for insect protection, herbicide resistance, virus resistance, enhanced nutrition, tolerance to environmental pressures and the production of edible vaccines. Most commercialised GMOs are insect resistant or herbicide tolerant crop plants. Genetically modified animals have been used for research, model animals and the production of agricultural or pharmaceutical products. The genetically modified animals include animals with genes knocked out, increased susceptibility to disease, hormones for extra growth and the ability to express proteins in their milk. has many applications to medicine that include the manufacturing of drugs, creation of model animals that mimic human conditions and gene therapy. One of the earliest uses of genetic engineering was to mass-produce human insulin in bacteria | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering This application has now been applied to, human growth hormones, follicle stimulating hormones (for treating infertility), human albumin, monoclonal antibodies, antihemophilic factors, vaccines and many other drugs. Mouse hybridomas, cells fused together to create monoclonal antibodies, have been adapted through genetic engineering to create human monoclonal antibodies. In 2017, genetic engineering of chimeric antigen receptors on a patient's own T-cells was approved by the U.S. FDA as a treatment for the cancer acute lymphoblastic leukemia. Genetically engineered viruses are being developed that can still confer immunity, but lack the infectious sequences. is also used to create animal models of human diseases. Genetically modified mice are the most common genetically engineered animal model. They have been used to study and model cancer (the oncomouse), obesity, heart disease, diabetes, arthritis, substance abuse, anxiety, aging and Parkinson disease. Potential cures can be tested against these mouse models. Also genetically modified pigs have been bred with the aim of increasing the success of pig to human organ transplantation. Gene therapy is the genetic engineering of humans, generally by replacing defective genes with effective ones. Clinical research using somatic gene therapy has been conducted with several diseases, including X-linked SCID, chronic lymphocytic leukemia (CLL), and Parkinson's disease. In 2012, Alipogene tiparvovec became the first gene therapy treatment to be approved for clinical use | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering In 2015 a virus was used to insert a healthy gene into the skin cells of a boy suffering from a rare skin disease, epidermolysis bullosa, in order to grow, and then graft healthy skin onto 80 percent of the boy's body which was affected by the illness. Germline gene therapy would result in any change being inheritable, which has raised concerns within the scientific community. In 2015, CRISPR was used to edit the DNA of non-viable human embryos, leading scientists of major world academies to call for a moratorium on inheritable human genome edits. There are also concerns that the technology could be used not just for treatment, but for enhancement, modification or alteration of a human beings' appearance, adaptability, intelligence, character or behavior. The distinction between cure and enhancement can also be difficult to establish. In November 2018, He Jiankui announced that he had edited the genomes of two human embryos, to attempt to disable the "CCR5" gene, which codes for a receptor that HIV uses to enter cells. He said that twin girls, Lulu and Nana, had been born a few weeks earlier. He said that the girls still carried functional copies of CCR5 along with disabled CCR5 (mosaicism) and were still vulnerable to HIV. The work was widely condemned as unethical, dangerous, and premature. Researchers are altering the genome of pigs to induce the growth of human organs to be used in transplants | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Scientists are creating "gene drives", changing the genomes of mosquitoes to make them immune to malaria, and then looking to spread the genetically altered mosquitoes throughout the mosquito population in the hopes of eliminating the disease. is an important tool for natural scientists, with the creation of transgenic organisms one of the most important tools for analysis of gene function. Genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research. Organisms are genetically engineered to discover the functions of certain genes. This could be the effect on the phenotype of the organism, where the gene is expressed or what other genes it interacts with. These experiments generally involve loss of function, gain of function, tracking and expression. Organisms can have their cells transformed with a gene coding for a useful protein, such as an enzyme, so that they will overexpress the desired protein. Mass quantities of the protein can then be manufactured by growing the transformed organism in bioreactor equipment using industrial fermentation, and then purifying the protein | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Some genes do not work well in bacteria, so yeast, insect cells or mammalians cells can also be used. These techniques are used to produce medicines such as insulin, human growth hormone, and vaccines, supplements such as tryptophan, aid in the production of food (chymosin in cheese making) and fuels. Other applications with genetically engineered bacteria could involve making them perform tasks outside their natural cycle, such as making biofuels, cleaning up oil spills, carbon and other toxic waste and detecting arsenic in drinking water. Certain genetically modified microbes can also be used in biomining and bioremediation, due to their ability to extract heavy metals from their environment and incorporate them into compounds that are more easily recoverable. In materials science, a genetically modified virus has been used in a research laboratory as a scaffold for assembling a more environmentally friendly lithium-ion battery. Bacteria have also been engineered to function as sensors by expressing a fluorescent protein under certain environmental conditions. One of the best-known and controversial applications of genetic engineering is the creation and use of genetically modified crops or genetically modified livestock to produce genetically modified food. Crops have been developed to increase production, increase tolerance to abiotic stresses, alter the composition of the food, or to produce novel products | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering The first crops to be released commercially on a large scale provided protection from insect pests or tolerance to herbicides. Fungal and virus resistant crops have also been developed or are in development. This makes the insect and weed management of crops easier and can indirectly increase crop yield. GM crops that directly improve yield by accelerating growth or making the plant more hardy (by improving salt, cold or drought tolerance) are also under development. In 2016 Salmon have been genetically modified with growth hormones to reach normal adult size much faster. GMOs have been developed that modify the quality of produce by increasing the nutritional value or providing more industrially useful qualities or quantities. The Amflora potato produces a more industrially useful blend of starches. Soybeans and canola have been genetically modified to produce more healthy oils. The first commercialised GM food was a tomato that had delayed ripening, increasing its shelf life. Plants and animals have been engineered to produce materials they do not normally make. Pharming uses crops and animals as bioreactors to produce vaccines, drug intermediates, or the drugs themselves; the useful product is purified from the harvest and then used in the standard pharmaceutical production process. Cows and goats have been engineered to express drugs and other proteins in their milk, and in 2009 the FDA approved a drug produced in goat milk. has potential applications in conservation and natural area management | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Gene transfer through viral vectors has been proposed as a means of controlling invasive species as well as vaccinating threatened fauna from disease. Transgenic trees have been suggested as a way to confer resistance to pathogens in wild populations. With the increasing risks of maladaptation in organisms as a result of climate change and other perturbations, facilitated adaptation through gene tweaking could be one solution to reducing extinction risks. Applications of genetic engineering in conservation are thus far mostly theoretical and have yet to be put into practice. is also being used to create microbial art. Some bacteria have been genetically engineered to create black and white photographs. Novelty items such as lavender-colored carnations, blue roses, and glowing fish have also been produced through genetic engineering. The regulation of genetic engineering concerns the approaches taken by governments to assess and manage the risks associated with the development and release of GMOs. The development of a regulatory framework began in 1975, at Asilomar, California. The Asilomar meeting recommended a set of voluntary guidelines regarding the use of recombinant technology. As the technology improved the US established a committee at the Office of Science and Technology, which assigned regulatory approval of GM food to the USDA, FDA and EPA. The Cartagena Protocol on Biosafety, an international treaty that governs the transfer, handling, and use of GMOs, was adopted on 29 January 2000 | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering One hundred and fifty-seven countries are members of the Protocol and many use it as a reference point for their own regulations. The legal and regulatory status of GM foods varies by country, with some nations banning or restricting them, and others permitting them with widely differing degrees of regulation. Some countries allow the import of GM food with authorisation, but either do not allow its cultivation (Russia, Norway, Israel) or have provisions for cultivation even though no GM products are yet produced (Japan, South Korea). Most countries that do not allow GMO cultivation do permit research. Some of the most marked differences occurring between the US and Europe. The US policy focuses on the product (not the process), only looks at verifiable scientific risks and uses the concept of substantial equivalence. The European Union by contrast has possibly the most stringent GMO regulations in the world. All GMOs, along with irradiated food, are considered "new food" and subject to extensive, case-by-case, science-based food evaluation by the European Food Safety Authority. The criteria for authorisation fall in four broad categories: "safety," "freedom of choice," "labelling," and "traceability." The level of regulation in other countries that cultivate GMOs lie in between Europe and the United States. One of the key issues concerning regulators is whether GM products should be labeled | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering The European Commission says that mandatory labeling and traceability are needed to allow for informed choice, avoid potential false advertising and facilitate the withdrawal of products if adverse effects on health or the environment are discovered. The American Medical Association and the American Association for the Advancement of Science say that absent scientific evidence of harm even voluntary labeling is misleading and will falsely alarm consumers. Labeling of GMO products in the marketplace is required in 64 countries. Labeling can be mandatory up to a threshold GM content level (which varies between countries) or voluntary. In Canada and the US labeling of GM food is voluntary, while in Europe all food (including processed food) or feed which contains greater than 0.9% of approved GMOs must be labelled. Critics have objected to the use of genetic engineering on several grounds, including ethical, ecological and economic concerns. Many of these concerns involve GM crops and whether food produced from them is safe and what impact growing them will have on the environment. These controversies have led to litigation, international trade disputes, and protests, and to restrictive regulation of commercial products in some countries. Accusations that scientists are "playing God" and other religious issues have been ascribed to the technology from the beginning | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering Other ethical issues raised include the patenting of life, the use of intellectual property rights, the level of labeling on products, control of the food supply and the objectivity of the regulatory process. Although doubts have been raised, economically most studies have found growing GM crops to be beneficial to farmers. Gene flow between GM crops and compatible plants, along with increased use of selective herbicides, can increase the risk of "superweeds" developing. Other environmental concerns involve potential impacts on non-target organisms, including soil microbes, and an increase in secondary and resistant insect pests. Many of the environmental impacts regarding GM crops may take many years to be understood and are also evident in conventional agriculture practices. With the commercialisation of genetically modified fish there are concerns over what the environmental consequences will be if they escape. There are three main concerns over the safety of genetically modified food: whether they may provoke an allergic reaction; whether the genes could transfer from the food into human cells; and whether the genes not approved for human consumption could outcross to other crops. There is a scientific consensus that currently available food derived from GM crops poses no greater risk to human health than conventional food, but that each GM food needs to be tested on a case-by-case basis before introduction. Nonetheless, members of the public are less likely than scientists to perceive GM foods as safe | https://en.wikipedia.org/wiki?curid=12383 |
Genetic engineering features in many science fiction stories. Frank Herbert's novel "The White Plague" described the deliberate use of genetic engineering to create a pathogen which specifically killed women. Another of Herbert's creations, the "Dune" series of novels, uses genetic engineering to create the powerful but despised Tleilaxu. Films such as "The Island" and "Blade Runner" bring the engineered creature to confront the person who created it or the being it was cloned from. Few films have informed audiences about genetic engineering, with the exception of the 1978 "The Boys from Brazil" and the 1993 "Jurassic Park", both of which made use of a lesson, a demonstration, and a clip of scientific film. methods are weakly represented in film; Michael Clark, writing for The Wellcome Trust, calls the portrayal of genetic engineering and biotechnology "seriously distorted" in films such as "The 6th Day". In Clark's view, the biotechnology is typically "given fantastic but visually arresting forms" while the science is either relegated to the background or fictionalised to suit a young audience. | https://en.wikipedia.org/wiki?curid=12383 |
Global Boundary Stratotype Section and Point A Global Boundary Stratotype Section and Point, abbreviated GSSP, is an internationally agreed upon reference point on a stratigraphic section which defines the lower boundary of a stage on the geologic time scale. The effort to define GSSPs is conducted by the International Commission on Stratigraphy, a part of the International Union of Geological Sciences. Most, but not all, GSSPs are based on paleontological changes. Hence GSSPs are usually described in terms of transitions between different faunal stages, though far more faunal stages have been described than GSSPs. The GSSP definition effort commenced in 1977. As of 2012, 64 of the 101 stages that need a GSSP have been formally defined. A geologic section has to fulfill a set of criteria to be adapted as a GSSP by the ICS. The following list summarizes the criteria: The Precambrian-Cambrian boundary GSSP at Fortune Head, Newfoundland is a typical GSSP. It is accessible by paved road and is set aside as a nature preserve. A continuous section is available from beds that are clearly Precambrian into beds that are clearly Cambrian. The boundary is set at the first appearance of a complex trace fossil "Treptichnus pedum" that is found worldwide. The Fortune Head GSSP is unlikely to be washed away or built over. Nonetheless, "Treptichnus pedum" is less than ideal as a marker fossil as it is not found in every Cambrian sequence, and it is not assured that it is found at the same level in every exposure | https://en.wikipedia.org/wiki?curid=12451 |
Global Boundary Stratotype Section and Point In fact, further eroding its value as a boundary marker, it has since been identified in strata 4m "below" the GSSP! However, no other fossil is known that would be preferable. There is no radiometrically datable bed at the boundary at Fortune Head, but there is one slightly above the boundary in similar beds nearby. These factors have led some geologists to suggest that this GSSP is in need of reassigning. Once a GSSP boundary has been agreed upon, a "golden spike" is driven into the geologic section to mark the precise boundary for future geologists (though in practice the "spike" need neither be golden nor an actual spike). The first stratigraphic boundary was defined in 1977 by identifying the Silurian-Devonian boundary with a bronze plaque at a locality called Klonk, northeast of the village of Suchomasty in the Czech Republic. GSSPs are also sometimes referred to as Golden Spikes. Because defining a GSSP depends on finding well-preserved geologic sections and identifying key events, this task becomes more difficult as one goes farther back in time. Before 630 million years ago, boundaries on the geologic timescale are defined simply by reference to fixed dates, known as "Global Standard Stratigraphic Ages". | https://en.wikipedia.org/wiki?curid=12451 |
Gene therapy (also called human gene transfer) is a medical field which focuses on the utilization of the therapeutic delivery of nucleic acid into a patient's cells as a drug to treat disease. The first attempt at modifying human DNA was performed in 1980 by Martin Cline, but the first successful nuclear gene transfer in humans, approved by the National Institutes of Health, was performed in May 1989. The first therapeutic use of gene transfer as well as the first direct insertion of human DNA into the nuclear genome was performed by French Anderson in a trial starting in September 1990. It is thought to be able to cure many genetic disorders or treat them over time. Between 1989 and December 2018, over 2,900 clinical trials were conducted, with more than half of them in phase I. As of 2017, Spark Therapeutics' Luxturna (RPE65 mutation-induced blindness) and Novartis' Kymriah (Chimeric antigen receptor T cell therapy) are the FDA's first approved gene therapies to enter the market. Since that time, drugs such as Novartis' Zolgensma and Alnylam's Patisiran have also received FDA approval, in addition to other companies' gene therapy drugs. Most of these approaches utilize adeno-associated viruses (AAVs) and lentiviruses for performing gene insertions, "in vivo" and "ex vivo", respectively. ASO / siRNA approaches such as those conducted by Alnylam and Ionis Pharmaceuticals require non-viral delivery systems, and utilize alternative mechanisms for trafficking to liver cells by way of GalNAc transporters | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The introduction of CRISPR gene editing has opened new doors for its application and utilization in gene therapy. Solutions to medical hurdles, such as the eradication of latent human immunodeficiency virus (HIV) reservoirs and correction of the mutation that causes sickle cell disease, may soon become a tangible reality. Not all medical procedures that introduce alterations to a patient's genetic makeup can be considered gene therapy. Bone marrow transplantation and organ transplants in general have been found to introduce foreign DNA into patients. is defined by the precision of the procedure and the intention of direct therapeutic effect. was conceptualized in 1972, by authors who urged caution before commencing human gene therapy studies. The first attempt, an unsuccessful one, at gene therapy (as well as the first case of medical transfer of foreign genes into humans not counting organ transplantation) was performed by Martin Cline on 10 July 1980. Cline claimed that one of the genes in his patients was active six months later, though he never published this data or had it verified and even if he is correct, it's unlikely it produced any significant beneficial effects treating beta-thalassemia. After extensive research on animals throughout the 1980s and a 1989 bacterial gene tagging trial on humans, the first gene therapy widely accepted as a success was demonstrated in a trial that started on 14 September 1990, when Ashi DeSilva was treated for ADA-SCID | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The first somatic treatment that produced a permanent genetic change was initiated in 1993. The goal was to cure malignant brain tumors by using recombinant DNA to transfer a gene making the tumor cells sensitive to a drug that in turn would cause the tumor cells to die. is a way to fix a genetic problem at its source. The polymers are either translated into proteins, interfere with target gene expression, or possibly correct genetic mutations. The most common form uses DNA that encodes a functional, therapeutic gene to replace a mutated gene. The polymer molecule is packaged within a "vector", which carries the molecule inside cells. Early clinical failures led to dismissals of gene therapy. Clinical successes since 2006 regained researchers' attention, although , it was still largely an experimental technique. These include treatment of retinal diseases Leber's congenital amaurosis and choroideremia, X-linked SCID, ADA-SCID, adrenoleukodystrophy, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), multiple myeloma, haemophilia, and Parkinson's disease. Between 2013 and April 2014, US companies invested over $600 million in the field. The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of certain cancers. In 2011 Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease, including critical limb ischemia | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In 2012 Glybera, a treatment for a rare inherited disorder, lipoprotein lipase deficiency became the first treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission. Following early advances in genetic engineering of bacteria, cells, and small animals, scientists started considering how to apply it to medicine. Two main approaches were considered – replacing or disrupting defective genes. Scientists focused on diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy, thalassemia, and sickle cell anemia. Glybera treats one such disease, caused by a defect in lipoprotein lipase. DNA must be administered, reach the damaged cells, enter the cell and either express or disrupt a protein. Multiple delivery techniques have been explored. The initial approach incorporated DNA into an engineered virus to deliver the DNA into a chromosome. Naked DNA approaches have also been explored, especially in the context of vaccine development. Generally, efforts focused on administering a gene that causes a needed protein to be expressed. More recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as zinc finger nucleases and CRISPR. The vector incorporates genes into chromosomes. The expressed nucleases then knock out and replace genes in the chromosome | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy these approaches involve removing cells from patients, editing a chromosome and returning the transformed cells to patients. Gene editing is a potential approach to alter the human genome to treat genetic diseases, viral diseases, and cancer. these approaches were still years from being medicine. may be classified into two types: In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Any such modifications affect the individual patient only, and are not inherited by offspring. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease. Over 600 clinical trials utilizing SCGT are underway in the US. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia, and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy. The complete correction of a genetic disorder or the replacement of multiple genes is not yet possible. Only a few of the trials are in the advanced stages. In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Australia, Canada, Germany, Israel, Switzerland, and the Netherlands prohibit GGT for application in human beings, for technical and ethical reasons, including insufficient knowledge about possible risks to future generations and higher risks versus SCGT. The US has no federal controls specifically addressing human genetic modification (beyond FDA regulations for therapies in general). The delivery of DNA into cells can be accomplished by multiple methods. The two major classes are recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or DNA complexes (non-viral methods). In order to replicate, viruses introduce their genetic material into the host cell, tricking the host's cellular machinery into using it as blueprints for viral proteins. Retroviruses go a stage further by having their genetic material copied into the genome of the host cell. Scientists exploit this by substituting a virus's genetic material with therapeutic DNA. (The term 'DNA' may be an oversimplification, as some viruses contain RNA, and gene therapy could take this form as well.) A number of viruses have been used for human gene therapy, including retroviruses, adenoviruses, herpes simplex, vaccinia, and adeno-associated virus. Like the genetic material (DNA or RNA) in viruses, therapeutic DNA can be designed to simply serve as a temporary blueprint that is degraded naturally or (at least theoretically) to enter the host's genome, becoming a permanent part of the host's DNA in infected cells | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Non-viral methods present certain advantages over viral methods, such as large scale production and low host immunogenicity. However, non-viral methods initially produced lower levels of transfection and gene expression, and thus lower therapeutic efficacy. Newer technologies offer promise of solving these problems, with the advent of increased cell-specific targeting and subcellular trafficking control. Methods for non-viral gene therapy include the injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles. More recent approaches, such as those performed by companies such as Ligandal, offer the possibility of creating cell-specific targeting technologies for a variety of gene therapy modalities, including RNA, DNA and gene editing tools such as CRISPR. Other companies, such as Arbutus Biopharma and Arcturus Therapeutics, offer non-viral, non-cell-targeted approaches that mainly exhibit liver trophism. In more recent years, startups such as Sixfold Bio, GenEdit, and Spotlight Therapeutics have begun to solve the non-viral gene delivery problem. Non-viral techniques offer the possibility of repeat dosing and greater tailorability of genetic payloads, which in the future will be more likely to take over viral-based delivery systems | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Companies such as Editas Medicine, Intellia Therapeutics, CRISPR Therapeutics, Casebia, Cellectis, Precision Biosciences, bluebird bio, and Sangamo have developed non-viral gene editing techniques, however frequently still use viruses for delivering gene insertion material following genomic cleavage by guided nucleases. These companies focus on gene editing, and still face major delivery hurdles. Moderna Therapeutics and CureVac focus on delivery of mRNA payloads, which are necessarily non-viral delivery problems. Alnylam, Dicerna Pharmaceuticals, and Ionis Pharmaceuticals focus on delivery of siRNA (antisense oligonucleotides) for gene suppression, which also necessitate non-viral delivery systems. In academic contexts, a number of laboratories are working on delivery of PEGylated particles, which form serum protein coronas and chiefly exhibit LDL receptor mediated uptake in cells "in vivo". Some of the unsolved problems include: Three patients' deaths have been reported in gene therapy trials, putting the field under close scrutiny. The first was that of Jesse Gelsinger, who died in 1999 because of immune rejection response. One X-SCID patient died of leukemia in 2003. In 2007, a rheumatoid arthritis patient died from an infection; the subsequent investigation concluded that the death was not related to gene therapy. However it is always important to remember that although deaths are rare they can still occur and it is very possible that certain types of gene therapy can cause certain cancers | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In 1972 Friedmann and Roblin authored a paper in "Science" titled "for human genetic disease?" Rogers (1970) was cited for proposing that "exogenous good DNA" be used to replace the defective DNA in those who suffer from genetic defects. In 1984 a retrovirus vector system was designed that could efficiently insert foreign genes into mammalian chromosomes. The first approved gene therapy clinical research in the US took place on 14 September 1990, at the National Institutes of Health (NIH), under the direction of William French Anderson. Four-year-old Ashanti DeSilva received treatment for a genetic defect that left her with ADA-SCID, a severe immune system deficiency. The defective gene of the patient's blood cells was replaced by the functional variant. Ashanti's immune system was partially restored by the therapy. Production of the missing enzyme was temporarily stimulated, but the new cells with functional genes were not generated. She led a normal life only with the regular injections performed every two months. The effects were successful, but temporary. Cancer gene therapy was introduced in 1992/93 (Trojan et al. 1993). The treatment of glioblastoma multiforme, the malignant brain tumor whose outcome is always fatal, was done using a vector expressing antisense IGF-I RNA (clinical trial approved by NIH protocol no.1602 24 November 1993, and by the FDA in 1994) | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy This therapy also represents the beginning of cancer immunogene therapy, a treatment which proves to be effective due to the anti-tumor mechanism of IGF-I antisense, which is related to strong immune and apoptotic phenomena. In 1992 Claudio Bordignon, working at the Vita-Salute San Raffaele University, performed the first gene therapy procedure using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases. In 2002 this work led to the publication of the first successful gene therapy treatment for adenosine deaminase deficiency (ADA-SCID). The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) from 2000 and 2002, was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the US, the United Kingdom, France, Italy, and Germany. In 1993 Andrew Gobea was born with SCID following prenatal genetic screening. Blood was removed from his mother's placenta and umbilical cord immediately after birth, to acquire stem cells. The allele that codes for adenosine deaminase (ADA) was obtained and inserted into a retrovirus. Retroviruses and stem cells were mixed, after which the viruses inserted the gene into the stem cell chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Injections of the ADA enzyme were also given weekly. For four years T cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed. Jesse Gelsinger's death in 1999 impeded gene therapy research in the US. As a result, the FDA suspended several clinical trials pending the reevaluation of ethical and procedural practices. The modified cancer gene therapy strategy of antisense IGF-I RNA (NIH n˚ 1602) using antisense / triple helix anti-IGF-I approach was registered in 2002 by Wiley gene therapy clinical trial - n˚ 635 and 636. The approach has shown promising results in the treatment of six different malignant tumors: glioblastoma, cancers of liver, colon, prostate, uterus, and ovary (Collaborative NATO Science Programme on Gene Therapy USA, France, Poland n˚ LST 980517 conducted by J. Trojan) (Trojan et al., 2012). This anti-gene antisense/triple helix therapy has proven to be efficient, due to the mechanism stopping simultaneously IGF-I expression on translation and transcription levels, strengthening anti-tumor immune and apoptotic phenomena. Sickle-cell disease can be treated in mice. The mice – which have essentially the same defect that causes human cases – used a viral vector to induce production of fetal hemoglobin (HbF), which normally ceases to be produced shortly after birth. In humans, the use of hydroxyurea to stimulate the production of HbF temporarily alleviates sickle cell symptoms | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The researchers demonstrated this treatment to be a more permanent means to increase therapeutic HbF production. A new gene therapy approach repaired errors in messenger RNA derived from defective genes. This technique has the potential to treat thalassaemia, cystic fibrosis and some cancers. Researchers created liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane. In 2003 a research team inserted genes into the brain for the first time. They used liposomes coated in a polymer called polyethylene glycol, which unlike viral vectors, are small enough to cross the blood–brain barrier. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced. Gendicine is a cancer gene therapy that delivers the tumor suppressor gene p53 using an engineered adenovirus. In 2003, it was approved in China for the treatment of head and neck squamous cell carcinoma. In March researchers announced the successful use of gene therapy to treat two adult patients for X-linked chronic granulomatous disease, a disease which affects myeloid cells and damages the immune system. The study is the first to show that gene therapy can treat the myeloid system. In May a team reported a way to prevent the immune system from rejecting a newly delivered gene | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Similar to organ transplantation, gene therapy has been plagued by this problem. The immune system normally recognizes the new gene as foreign and rejects the cells carrying it. The research utilized a newly uncovered network of genes regulated by molecules known as microRNAs. This natural function selectively obscured their therapeutic gene in immune system cells and protected it from discovery. Mice infected with the gene containing an immune-cell microRNA target sequence did not reject the gene. In August scientists successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. In November researchers reported on the use of VRX496, a gene-based immunotherapy for the treatment of HIV that uses a lentiviral vector to deliver an antisense gene against the HIV envelope. In a phase I clinical trial, five subjects with chronic HIV infection who had failed to respond to at least two antiretroviral regimens were treated. A single intravenous infusion of autologous CD4 T cells genetically modified with VRX496 was well tolerated. All patients had stable or decreased viral load; four of the five patients had stable or increased CD4 T cell counts. All five patients had stable or increased immune response to HIV antigens and other pathogens. This was the first evaluation of a lentiviral vector administered in a US human clinical trial. In May researchers announced the first gene therapy trial for inherited retinal disease | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The first operation was carried out on a 23-year-old British male, Robert Johnson, in early 2007. Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of a small clinical trial in children were published in April. Delivery of recombinant adeno-associated virus (AAV) carrying RPE65 yielded positive results. In May two more groups reported positive results in independent clinical trials using gene therapy to treat the condition. In all three clinical trials, patients recovered functional vision without apparent side-effects. In September researchers were able to give trichromatic vision to squirrel monkeys. In November 2009, researchers halted a fatal genetic disorder called adrenoleukodystrophy in two children using a lentivirus vector to deliver a functioning version of ABCD1, the gene that is mutated in the disorder. An April paper reported that gene therapy addressed achromatopsia (color blindness) in dogs by targeting cone photoreceptors. Cone function and day vision were restored for at least 33 months in two young specimens. The therapy was less efficient for older dogs. In September it was announced that an 18-year-old male patient in France with beta-thalassemia major had been successfully treated. Beta-thalassemia major is an inherited blood disease in which beta haemoglobin is missing and patients are dependent on regular lifelong blood transfusions | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The technique used a lentiviral vector to transduce the human ß-globin gene into purified blood and marrow cells obtained from the patient in June 2007. The patient's haemoglobin levels were stable at 9 to 10 g/dL. About a third of the hemoglobin contained the form introduced by the viral vector and blood transfusions were not needed. Further clinical trials were planned. Bone marrow transplants are the only cure for thalassemia, but 75% of patients do not find a matching donor. Cancer immunogene therapy using modified antigene, antisense/triple helix approach was introduced in South America in 2010/11 in La Sabana University, Bogota (Ethical Committee 14 December 2010, no P-004-10). Considering the ethical aspect of gene diagnostic and gene therapy targeting IGF-I, the IGF-I expressing tumors i.e. lung and epidermis cancers were treated (Trojan et al. 2016). In 2007 and 2008, a man (Timothy Ray Brown) was cured of HIV by repeated hematopoietic stem cell transplantation (see also allogeneic stem cell transplantation, allogeneic bone marrow transplantation, allotransplantation) with double-delta-32 mutation which disables the CCR5 receptor. This cure was accepted by the medical community in 2011. It required complete ablation of existing bone marrow, which is very debilitating. In August two of three subjects of a pilot study were confirmed to have been cured from chronic lymphocytic leukemia (CLL) | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy The therapy used genetically modified T cells to attack cells that expressed the CD19 protein to fight the disease. In 2013, the researchers announced that 26 of 59 patients had achieved complete remission and the original patient had remained tumor-free. Human HGF plasmid DNA therapy of cardiomyocytes is being examined as a potential treatment for coronary artery disease as well as treatment for the damage that occurs to the heart after myocardial infarction. In 2011 Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease, including critical limb ischemia; it delivers the gene encoding for VEGF. Neovasculogen is a plasmid encoding the CMV promoter and the 165 amino acid form of VEGF. The FDA approved Phase 1 clinical trials on thalassemia major patients in the US for 10 participants in July. The study was expected to continue until 2015. In July 2012, the European Medicines Agency recommended approval of a gene therapy treatment for the first time in either Europe or the United States. The treatment used Alipogene tiparvovec (Glybera) to compensate for lipoprotein lipase deficiency, which can cause severe pancreatitis. The recommendation was endorsed by the European Commission in November 2012 and commercial rollout began in late 2014. Alipogene tiparvovec was expected to cost around $1.6 million per treatment in 2012, revised to $1 million in 2015, making it the most expensive medicine in the world at the time | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy , only the patients treated in clinical trials and a patient who paid the full price for treatment have received the drug. In December 2012, it was reported that 10 of 13 patients with multiple myeloma were in remission "or very close to it" three months after being injected with a treatment involving genetically engineered T cells to target proteins NY-ESO-1 and LAGE-1, which exist only on cancerous myeloma cells. In March researchers reported that three of five adult subjects who had acute lymphocytic leukemia (ALL) had been in remission for five months to two years after being treated with genetically modified T cells which attacked cells with CD19 genes on their surface, i.e. all B-cells, cancerous or not. The researchers believed that the patients' immune systems would make normal T-cells and B-cells after a couple of months. They were also given bone marrow. One patient relapsed and died and one died of a blood clot unrelated to the disease. Following encouraging Phase 1 trials, in April, researchers announced they were starting Phase 2 clinical trials (called CUPID2 and SERCA-LVAD) on 250 patients at several hospitals to combat heart disease. The therapy was designed to increase the levels of SERCA2, a protein in heart muscles, improving muscle function. The FDA granted this a Breakthrough Therapy Designation to accelerate the trial and approval process. In 2016 it was reported that no improvement was found from the CUPID 2 trial | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In July researchers reported promising results for six children with two severe hereditary diseases had been treated with a partially deactivated lentivirus to replace a faulty gene and after 7–32 months. Three of the children had metachromatic leukodystrophy, which causes children to lose cognitive and motor skills. The other children had Wiskott–Aldrich syndrome, which leaves them to open to infection, autoimmune diseases, and cancer. Follow up trials with gene therapy on another six children with Wiskott–Aldrich syndrome were also reported as promising. In October researchers reported that two children born with adenosine deaminase severe combined immunodeficiency disease (ADA-SCID) had been treated with genetically engineered stem cells 18 months previously and that their immune systems were showing signs of full recovery. Another three children were making progress. In 2014 a further 18 children with ADA-SCID were cured by gene therapy. ADA-SCID children have no functioning immune system and are sometimes known as "bubble children." Also in October researchers reported that they had treated six hemophilia sufferers in early 2011 using an adeno-associated virus. Over two years later all six were producing clotting factor. In January researchers reported that six choroideremia patients had been treated with adeno-associated virus with a copy of REP1. Over a six-month to two-year period all had improved their sight | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy By 2016, 32 patients had been treated with positive results and researchers were hopeful the treatment would be long-lasting. Choroideremia is an inherited genetic eye disease with no approved treatment, leading to loss of sight. In March researchers reported that 12 HIV patients had been treated since 2009 in a trial with a genetically engineered virus with a rare mutation (CCR5 deficiency) known to protect against HIV with promising results. Clinical trials of gene therapy for sickle cell disease were started in 2014. In February LentiGlobin BB305, a gene therapy treatment undergoing clinical trials for treatment of beta thalassemia gained FDA "breakthrough" status after several patients were able to forgo the frequent blood transfusions usually required to treat the disease. In March researchers delivered a recombinant gene encoding a broadly neutralizing antibody into monkeys infected with simian HIV; the monkeys' cells produced the antibody, which cleared them of HIV. The technique is named immunoprophylaxis by gene transfer (IGT). Animal tests for antibodies to ebola, malaria, influenza, and hepatitis were underway. In March, scientists, including an inventor of CRISPR, Jennifer Doudna, urged a worldwide moratorium on germline gene therapy, writing "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations" | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In October, researchers announced that they had treated a baby girl, Layla Richards, with an experimental treatment using donor T-cells genetically engineered using TALEN to attack cancer cells. One year after the treatment she was still free of her cancer (a highly aggressive form of acute lymphoblastic leukaemia [ALL]). Children with highly aggressive ALL normally have a very poor prognosis and Layla's disease had been regarded as terminal before the treatment. In December, scientists of major world academies called for a moratorium on inheritable human genome edits, including those related to CRISPR-Cas9 technologies but that basic research including embryo gene editing should continue. In April the Committee for Medicinal Products for Human Use of the European Medicines Agency endorsed a gene therapy treatment called Strimvelis and the European Commission approved it in June. This treats children born with adenosine deaminase deficiency and who have no functioning immune system. This was the second gene therapy treatment to be approved in Europe. In October, Chinese scientists reported they had started a trial to genetically modify T-cells from 10 adult patients with lung cancer and reinject the modified T-cells back into their bodies to attack the cancer cells. The T-cells had the PD-1 protein (which stops or slows the immune response) removed using CRISPR-Cas9 | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy A 2016 Cochrane systematic review looking at data from four trials on topical cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy does not support its clinical use as a mist inhaled into the lungs to treat cystic fibrosis patients with lung infections. One of the four trials did find weak evidence that liposome-based CFTR gene transfer therapy may lead to a small respiratory improvement for people with CF. This weak evidence is not enough to make a clinical recommendation for routine CFTR gene therapy. In February Kite Pharma announced results from a clinical trial of CAR-T cells in around a hundred people with advanced Non-Hodgkin lymphoma. In March, French scientists reported on clinical research of gene therapy to treat sickle-cell disease. In August, the FDA approved tisagenlecleucel for acute lymphoblastic leukemia. Tisagenlecleucel is an adoptive cell transfer therapy for B-cell acute lymphoblastic leukemia; T cells from a person with cancer are removed, genetically engineered to make a specific T-cell receptor (a chimeric T cell receptor, or "CAR-T") that reacts to the cancer, and are administered back to the person. The T cells are engineered to target a protein called CD19 that is common on B cells. This is the first form of gene therapy to be approved in the United States. In October, a similar therapy called axicabtagene ciloleucel was approved for non-Hodgkin lymphoma | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In December the results of using an adeno-associated virus with blood clotting factor VIII to treat nine haemophilia A patients were published. Six of the seven patients on the high dose regime increased the level of the blood clotting VIII to normal levels. The low and medium dose regimes had no effect on the patient's blood clotting levels. In December, the FDA approved Luxturna, the first "in vivo" gene therapy, for the treatment of blindness due to Leber's congenital amaurosis. The price of this treatment was 850,000 US dollars for both eyes. A need was identified for high quality randomised controlled trials assessing the risks and benefits involved with gene therapy for people with sickle cell disease. In February, medical scientists working with Sangamo Therapeutics, headquartered in Richmond, California, announced the first ever "in body" human gene editing therapy to permanently alter DNA - in a patient with Hunter syndrome. Clinical trials by Sangamo involving gene editing using Zinc Finger Nuclease (ZFN) are ongoing. In May, the FDA approved Zolgensma for treating spinal muscular atrophy in children under 2 years. The list price of Zolgensma was set at $2.125 million per dose, making it the most expensive drug ever. In June, the EMA approved Zynteglo for treating beta thalassemia for patients 12 years or older. In July, Allergan and Editas Medicine announced phase 1/2 clinical trial of AGN-151587 for the treatment of Leber congenital amaurosis 10 | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy It will be the world's first "in vivo" study of a CRISPR-based human gene editing therapy, where the editing takes place inside the human body. Speculated uses for gene therapy include: Athletes might adopt gene therapy technologies to improve their performance. Gene doping is not known to occur, but multiple gene therapies may have such effects. Kayser et al. argue that gene doping could level the playing field if all athletes receive equal access. Critics claim that any therapeutic intervention for non-therapeutic/enhancement purposes compromises the ethical foundations of medicine and sports. Genetic engineering could be used to cure diseases, but also to change physical appearance, metabolism, and even improve physical capabilities and mental faculties such as memory and intelligence. Ethical claims about germline engineering include beliefs that every fetus has a right to remain genetically unmodified, that parents hold the right to genetically modify their offspring, and that every child has the right to be born free of preventable diseases. For parents, genetic engineering could be seen as another child enhancement technique to add to diet, exercise, education, training, cosmetics, and plastic surgery. Another theorist claims that moral concerns limit but do not prohibit germline engineering. A recent issue of the journal "Bioethics" was devoted to moral issues surrounding germline genetic engineering in people | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy Possible regulatory schemes include a complete ban, provision to everyone, or professional self-regulation. The American Medical Association’s Council on Ethical and Judicial Affairs stated that "genetic interventions to enhance traits should be considered permissible only in severely restricted situations: (1) clear and meaningful benefits to the fetus or child; (2) no trade-off with other characteristics or traits; and (3) equal access to the genetic technology, irrespective of income or other socioeconomic characteristics." As early in the history of biotechnology as 1990, there have been scientists opposed to attempts to modify the human germline using these new tools, and such concerns have continued as technology progressed. With the advent of new techniques like CRISPR, in March 2015 a group of scientists urged a worldwide moratorium on clinical use of gene editing technologies to edit the human genome in a way that can be inherited. In April 2015, researchers sparked controversy when they reported results of basic research to edit the DNA of non-viable human embryos using CRISPR. A committee of the American National Academy of Sciences and National Academy of Medicine gave qualified support to human genome editing in 2017 once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight." Regulations covering genetic modification are part of general guidelines about human-involved biomedical research | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy There are no international treaties which are legally binding in this area, but there are recommendations for national laws from various bodies. The Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects) was amended by the World Medical Association's General Assembly in 2008. This document provides principles physicians and researchers must consider when involving humans as research subjects. The Statement on Gene Therapy Research initiated by the Human Genome Organization (HUGO) in 2001 provides a legal baseline for all countries. HUGO's document emphasizes human freedom and adherence to human rights, and offers recommendations for somatic gene therapy, including the importance of recognizing public concerns about such research. No federal legislation lays out protocols or restrictions about human genetic engineering. This subject is governed by overlapping regulations from local and federal agencies, including the Department of Health and Human Services, the FDA and NIH's Recombinant DNA Advisory Committee. Researchers seeking federal funds for an investigational new drug application, (commonly the case for somatic human genetic engineering,) must obey international and federal guidelines for the protection of human subjects. NIH serves as the main gene therapy regulator for federally funded research. Privately funded research is advised to follow these regulations | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy NIH provides funding for research that develops or enhances genetic engineering techniques and to evaluate the ethics and quality in current research. The NIH maintains a mandatory registry of human genetic engineering research protocols that includes all federally funded projects. An NIH advisory committee published a set of guidelines on gene manipulation. The guidelines discuss lab safety as well as human test subjects and various experimental types that involve genetic changes. Several sections specifically pertain to human genetic engineering, including Section III-C-1. This section describes required review processes and other aspects when seeking approval to begin clinical research involving genetic transfer into a human patient. The protocol for a gene therapy clinical trial must be approved by the NIH's Recombinant DNA Advisory Committee prior to any clinical trial beginning; this is different from any other kind of clinical trial. As with other kinds of drugs, the FDA regulates the quality and safety of gene therapy products and supervises how these products are used clinically. Therapeutic alteration of the human genome falls under the same regulatory requirements as any other medical treatment. Research involving human subjects, such as clinical trials, must be reviewed and approved by the FDA and an Institutional Review Board. is the basis for the plotline of the film "I Am Legend" and the TV show "Will Gene Therapy Change the Human Race?" | https://en.wikipedia.org/wiki?curid=12891 |
Gene therapy In 1994, gene therapy was a plot element in "The Erlenmeyer Flask", the first-season finale of "The X-Files"; it is also used in "Stargate" as a means of allowing humans to use Ancient technology. | https://en.wikipedia.org/wiki?curid=12891 |
Outline of health sciences The following outline is provided as an overview of and topical guide to health sciences: Health sciences – are those sciences which focus on health, or health care, as core parts of their subject matter. Because these two subject matter relate to multiple academic disciplines, both STEM disciplines as well as emerging patient safety disciplines (such as social care research) are relevant to current health scientific knowledge. Health sciences knowledge bases are currently diverse, with intellectual foundations which are sometimes mutually-inconsistent. There is currently an existing bias in the field, towards high valuation of knowledge deriving from controlling views on human agency (as epitomized by the epistemological basis of Randomized Control Trial designs); compare this against the more naturalistic views on human agency taken by research based on Ethnography for example). Mental health Social health Physical health Medicine – applied science or practice of the diagnosis, treatment, and prevention of disease. It encompasses a variety of health care practices evolved to maintain and restore health by the prevention and treatment of illness. Some of its branches are: | https://en.wikipedia.org/wiki?curid=14002 |
Human cloning is the creation of a genetically identical copy (or clone) of a human. The term is generally used to refer to artificial human cloning, which is the reproduction of human cells and tissue. It does not refer to the natural conception and delivery of identical twins. The possibility of person cloning has raised controversies. These ethical concerns have prompted several nations to pass laws regarding human cloning and its legality. Two commonly discussed types of theoretical human cloning are "therapeutic cloning" and "reproductive cloning". Therapeutic cloning would involve cloning cells from a human for use in medicine and transplants, and is an active area of research, but is not in medical practice anywhere in the world, . Two common methods of therapeutic cloning that are being researched are somatic-cell nuclear transfer and, more recently, pluripotent stem cell induction. Reproductive cloning would involve making an entire cloned human, instead of just specific cells or tissues. Although the possibility of cloning humans had been the subject of speculation for much of the 20th century, scientists and policymakers began to take the prospect seriously in 1969. J. B. S. Haldane was the first to introduce the idea of human cloning, for which he used the terms "clone" and "cloning", which had been used in agriculture since the early 20th century | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning In his speech on "Biological Possibilities for the Human Species of the Next Ten Thousand Years" at the "Ciba Foundation Symposium on Man and his Future" in 1963, he said: Nobel Prize-winning geneticist Joshua Lederberg advocated cloning and genetic engineering in an article in "The American Naturalist" in 1966 and again, the following year, in "The Washington Post". He sparked a debate with conservative bioethicist Leon Kass, who wrote at the time that "the programmed reproduction of man will, in fact, dehumanize him." Another Nobel Laureate, James D. Watson, publicized the potential and the perils of cloning in his "Atlantic Monthly" essay, "Moving Toward the Clonal Man", in 1971. With the cloning of a sheep known as Dolly in 1996 by somatic cell nuclear transfer (SCNT), the idea of human cloning became a hot debate topic. Many nations outlawed it, while a few scientists promised to make a clone within the next few years. The first hybrid human clone was created in November 1998, by Advanced Cell Technology. It was created using SCNT; a nucleus was taken from a man's leg cell and inserted into a cow's egg from which the nucleus had been removed, and the hybrid cell was cultured and developed into an embryo. The embryo was destroyed after 12 days. In 2004 and 2005, Hwang Woo-suk, a professor at Seoul National University, published two separate articles in the journal " Science" claiming to have successfully harvested pluripotent, embryonic stem cells from a cloned human blastocyst using SCNT techniques | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning Hwang claimed to have created eleven different patient-specific stem cell lines. This would have been the first major breakthrough in human cloning. However, in 2006 "Science" retracted both of his articles on clear evidence that much of his data from the experiments was fabricated. In January 2008, Dr. Andrew French and Samuel Wood of the biotechnology company Stemagen announced that they successfully created the first five mature human embryos using SCNT. In this case, each embryo was created by taking a nucleus from a skin cell (donated by Wood and a colleague) and inserting it into a human egg from which the nucleus had been removed. The embryos were developed only to the blastocyst stage, at which point they were studied in processes that destroyed them. Members of the lab said that their next set of experiments would aim to generate embryonic stem cell lines; these are the "holy grail" that would be useful for therapeutic or reproductive cloning. In 2011, scientists at the New York Stem Cell Foundation announced that they had succeeded in generating embryonic stem cell lines, but their process involved leaving the oocyte's nucleus in place, resulting in triploid cells, which would not be useful for cloning. In 2013, a group of scientists led by Shoukhrat Mitalipov published the first report of embryonic stem cells created using SCNT. In this experiment, the researchers developed a protocol for using SCNT in human cells, which differs slightly from the one used in other organisms | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning Four embryonic stem cell lines from human fetal somatic cells were derived from those blastocysts. All four lines were derived using oocytes from the same donor, ensuring that all mitochondrial DNA inherited was identical. A year later, a team led by Robert Lanza at Advanced Cell Technology reported that they had replicated Mitalipov's results and further demonstrated the effectiveness by cloning adult cells using SCNT. In 2018, the first successful cloning of primates using SCNT was reported with the birth of two live female clones, crab-eating macaques named Zhong Zhong and Hua Hua. In somatic cell nuclear transfer ("SCNT"), the nucleus of a somatic cell is taken from a donor and transplanted into a host egg cell, which had its own genetic material removed previously, making it an enucleated egg. After the donor somatic cell genetic material is transferred into the host oocyte with a micropipette, the somatic cell genetic material is fused with the egg using an electric current. Once the two cells have fused, the new cell can be permitted to grow in a surrogate or artificially. This is the process that was used to successfully clone Dolly the sheep (see section on History in this article) | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning The technique, now refined, has indicated that it was possible to replicate cells and reestablish pluripotency-"the potential of an embryonic cell to grow into any one of the numerous different types of mature body cells that make up a complete organism" Creating induced pluripotent stem cells ("iPSCs") is a long and inefficient process. Pluripotency refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous tissue). A specific set of genes, often called "reprogramming factors", are introduced into a specific adult cell type. These factors send signals in the mature cell that cause the cell to become a pluripotent stem cell. This process is highly studied and new techniques are being discovered frequently on how to better this induction process. Depending on the method used, reprogramming of adult cells into iPSCs for implantation could have severe limitations in humans. If a virus is used as a reprogramming factor for the cell, cancer-causing genes called oncogenes may be activated. These cells would appear as rapidly dividing cancer cells that do not respond to the body's natural cell signaling process. However, in 2008 scientists discovered a technique that could remove the presence of these oncogenes after pluripotency induction, thereby increasing the potential use of iPSC in humans | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning Both the processes of SCNT and iPSCs have benefits and deficiencies. Historically, reprogramming methods were better studied than SCNT derived embryonic stem cells (ESCs). However, more recent studies have put more emphasis on developing new procedures for SCNT-ESCs. The major advantage of SCNT over iPSCs at this time is the speed with which cells can be produced. iPSCs derivation takes several months while SCNT would take a much shorter time, which could be important for medical applications. New studies are working to improve the process of iPSC in terms of both speed and efficiency with the discovery of new reprogramming factors in oocytes. Another advantage SCNT could have over iPSCs is its potential to treat mitochondrial disease, as it utilizes a donor oocyte. No other advantages are known at this time in using stem cells derived from one method over stem cells derived from the other. Work on cloning techniques has advanced our basic understanding of developmental biology in humans. Observing human pluripotent stem cells grown in culture provides great insight into human embryo development, which otherwise cannot be seen. Scientists are now able to better define steps of early human development. Studying signal transduction along with genetic manipulation within the early human embryo has the potential to provide answers to many developmental diseases and defects. Many human-specific signaling pathways have been discovered by studying human embryonic stem cells | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning Studying developmental pathways in humans has given developmental biologists more evidence toward the hypothesis that developmental pathways are conserved throughout species. iPSCs and cells created by SCNT are useful for research into the causes of disease, and as model systems used in drug discovery. Cells produced with SCNT, or iPSCs could eventually be used in stem cell therapy, or to create organs to be used in transplantation, known as regenerative medicine. Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplantation is a widely used form of stem cell therapy. No other forms of stem cell therapy are in clinical use at this time. Research is underway to potentially use stem cell therapy to treat heart disease, diabetes, and spinal cord injuries. Regenerative medicine is not in clinical practice, but is heavily researched for its potential uses. This type of medicine would allow for autologous transplantation, thus removing the risk of organ transplant rejection by the recipient. For instance, a person with liver disease could potentially have a new liver grown using their same genetic material and transplanted to remove the damaged liver. In current research, human pluripotent stem cells have been promised as a reliable source for generating human neurons, showing the potential for regenerative medicine in brain and neural injuries | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning In bioethics, the ethics of cloning refers to a variety of ethical positions regarding the practice and possibilities of cloning, especially human cloning. While many of these views are religious in origin, the questions raised by cloning are faced by secular perspectives as well. Human therapeutic and reproductive cloning are not commercially used; animals are currently cloned in laboratories and in livestock production. Advocates support development of therapeutic cloning in order to generate tissues and whole organs to treat patients who otherwise cannot obtain transplants, to avoid the need for immunosuppressive drugs, and to stave off the effects of aging. Advocates for reproductive cloning believe that parents who cannot otherwise procreate should have access to the technology. Opposition to therapeutic cloning mainly centers around the status of embryonic stem cells, which has connections with the abortion debate. Some opponents of reproductive cloning have concerns that technology is not yet developed enough to be safe – for example, the position of the American Association for the Advancement of Science , while others emphasize that reproductive cloning could be prone to abuse (leading to the generation of humans whose organs and tissues would be harvested), and have concerns about how cloned individuals could integrate with families and with society at large. Some opponents will raise questions on whether clones have rights | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning "Cloning's Future" raises series question on whether the embryo's have any rights or if the donor outweigh's the right of the embryo to live. Religious groups are divided, with some opposing the technology as usurping God's role in creation and, to the extent embryos are used, destroying a human life; others support therapeutic cloning's potential life-saving benefits. In 2018 it was reported that about 70 countries had banned human cloning. is banned by the Presidential Decree 200/97 of 7 March 1997. Australia has prohibited human cloning, though , a bill legalizing therapeutic cloning and the creation of human embryos for stem cell research passed the House of Representatives. Within certain regulatory limits, and subject to the effect of state legislation, therapeutic cloning is now legal in some parts of Australia. Canadian law prohibits the following: cloning humans, cloning stem cells, growing human embryos for research purposes, and buying or selling of embryos, sperm, eggs or other human reproductive material. It also bans making changes to human DNA that would pass from one generation to the next, including use of animal DNA in humans. Surrogate mothers are legally allowed, as is donation of sperm or eggs for reproductive purposes. Human embryos and stem cells are also permitted to be donated for research. There have been consistent calls in Canada to ban human reproductive cloning since the 1993 Report of the Royal Commission on New Reproductive Technologies | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning Polls have indicated that an overwhelming majority of Canadians oppose human reproductive cloning, though the regulation of human cloning continues to be a significant national and international policy issue. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. is prohibited in Article 133 of the Colombian Penal Code. The European Convention on Human Rights and Biomedicine prohibits human cloning in one of its additional protocols, this protocol has been ratified by 25 states. The Charter of Fundamental Rights of the European Union explicitly prohibits reproductive human cloning. The charter is legally binding for the institutions of the European Union under the Treaty of Lisbon and for some member countries of the Union implementing EU regulations. India does not have specific law regarding cloning but has guidelines prohibiting whole human cloning or reproductive cloning. India allows therapeutic cloning and the use of embryonic stem cells for research purposes. forbidden by article 87 of Act of 25 June 2015. The Federal Assembly of Russia introduced the Federal Law N 54-FZ "On the temporary ban on human cloning" in April 19, 2002. On May 20, 2002 President Vladimir Putin signed this moratorium on the implementation of human cloning. On March 29, 2010 The Federal Assembly introduced second revision of this law without time limit | https://en.wikipedia.org/wiki?curid=14094 |
Human cloning is explicitly prohibited in Article 24, "Right to Life" of the 2006 Constitution of Serbia. In terms of section 39A of the Human Tissue Act 65 of 1983, genetic manipulation of gametes or zygotes outside the human body is absolutely prohibited. A zygote is the cell resulting from the fusion of two gametes; thus the fertilised ovum. Section 39A thus prohibits human cloning. On January 14, 2001 the British government passed The Human Fertilisation and Embryology (Research Purposes) Regulations 2001 to amend the Human Fertilisation and Embryology Act 1990 by extending allowable reasons for embryo research to permit research around stem cells and cell nuclear replacement, thus allowing therapeutic cloning. However, on November 15, 2001, a pro-life group won a High Court legal challenge, which struck down the regulation and effectively left all forms of cloning unregulated in the UK. Their hope was that Parliament would fill this gap by passing prohibitive legislation. Parliament was quick to pass the Human Reproductive Cloning Act 2001 which explicitly prohibited reproductive cloning. The remaining gap with regard to therapeutic cloning was closed when the appeals courts reversed the previous decision of the High Court. The first license was granted on August 11, 2004 to researchers at the University of Newcastle to allow them to investigate treatments for diabetes, Parkinson's disease and Alzheimer's disease | https://en.wikipedia.org/wiki?curid=14094 |
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