History of genetic engineering

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Genetic modification caused by human activity has been occurring since humans first domesticated organisms in 12 000 BC. Genetic engineering as the direct transfer of DNA from one organism to another was first accomplished by Herbert Boyer and Stanley Cohen in 1973. Advances have allowed scientists to manipulate and add genes to a variety of different organism and to induce a range of different effects. Since 1976 the technology has been commercialised, with companies producing and selling genetically modified food and medicine.

Agriculture[edit]

The first human manipulation of genes occurred during the domestication of plants and animals through artificial selection. The dog is believed to be the first animal domesticated, most likely arising from the grey wolf, with fossil evidence dating to about 12,000 BC.[1]:1 The other carnivores domesticated in prehistoric times were the cat and polecat.[1]:2 Sheep and goats were domesticated around 8,000 BC in the Fertile Crescent, while pigs appeared in China about 7 000 BC, yaks in Tibet about 5,000 BC and horses in Eastern Europe around 4,000 BC.[1]:3 The first domesticated bird was the rock pigeon, appearing in Greece, Egypt and Mesopotamia around 3 000 BC [1]:4 and the first domesticated fish was probably carp, raised as food in China around 1,000 BC.[1]:5

The first evidence of plant domestication comes from emmer and einkorn wheat found in pre-Pottery Neolithic A villages in Southwest Asia dated about 10,500 to 10,100 BC.[2]:1 The Fertile Crescent of Western Asia, Egypt, and India were sites of the earliest planned sowing and harvesting of plants that had previously been gathered in the wild. Independent development of agriculture occurred in northern and southern China, Africa's Sahel, New Guinea and several regions of the Americas.[3] The eight Neolithic founder crops (emmer wheat, einkorn wheat, barley, peas, lentils, bitter vetch, chick peas and flax) had all appeared by about 7000 BC.[4] Horticulture first appears in the Levant during the Chalcolithic period about 6 800 to 6,300 BC.[2]:5 Due to the soft tissues, archeological evidence for early vegetables is scarce. The earliest vegetable remains have been found in Egyptian caves that date back to the 2nd millennium BC.[2]:6

Selective breeding of domesticated plants was once the main way early farmers shaped organisms to suit their needs. Charles Darwin described three types of selection: methodical (when selecting for some pre-determined characteristic), unconscious (when a characteristic is selected simply because it is desirable), and natural (when a trait that helps an organism survive better is passed on).[5]:25 Early breeding relied on unconscious and natural selection. The introduction of methodical selection is unknown.[5]:25 Common characteristics that were bred into domesticated plants include grains that did not shatter to allow easier harvesting, uniform ripening, shorter lifespans that translate to faster growing, loss of toxic compounds, and productivity.[5]:27-30 Some plants, like the Banana, were able to be propagated by vegetative cloning. Offspring often did not contain seeds, and therefore sterile. However, these offspring were usually juicier and larger. Propagation through cloning allows these mutant varieties to be cultivated despite their lack of seeds.[5]:31

Hybridization was another way that rapid changes in plant's makeup were introduced. It often increased vigor in plants, and combined desirable traits together. Hybridization most likely first occurred when humans first grew similar, yet slightly different plants in close proximity.[5]:32 Triticum aestivum, wheat used in baking bread, is an allopolyploid. Its creation is the result of two separate hybridization events.[6]

X-rays were first used to deliberately mutate plants in 1927. Between 1927 and 2007, more than 2,540 genetically mutated plant varieties had been produced using x-rays.[7]

Genetics[edit]

Main article: History of genetics

Various genetic discoveries have been essential in the development of genetic engineering. Genetic inheritance was first discovered by Gregor Mendel in 1865 following experiments crossing peas. Although largely ignored for 34 years he provided the first evidence of hereditary segregation and independent assortment.[8] In 1889 Hugo de Vries came up with the name "(pan)gene" for after postulating that particles are responsible for inheritance of characteristics[9] and the term "genetics" was coined by William Bateson in 1905.[10] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which Avery, MacLeod and McCarty later (1944) identified as DNA. Edward Lawrie Tatum and George Wells Beadle developed the central dogma that genes code for proteins in 1941. The double helix structure of DNA was identified by James Watson and Francis Crick in 1953.

As well as discovering how DNA works, tools had to be developed that allowed it to be manipulated. In 1970 Hamilton Smiths lab discovered restriction enzymes that allowed DNA to be cut at specific places and separated out on an electrophoresis gel. This enabled scientists to isolate genes from an organism's genome.[11] DNA ligases, that join broken DNA together, had been discovered earlier in 1967[12] and by combining the two enzymes it was possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952,[13] became important tools for transferring information between cells and replicating DNA sequences. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers. Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified and aided identification and isolation of genetic material.

As well as manipulating the DNA techniques had to be developed for its insertion (known as transformation) into an organism's genome. Griffiths experiment had already shown that some bacteria had the ability to naturally uptake and express foreign DNA. Artificial competence was induced in Escherichia coli in 1970 when Morton Mandel and Akiko Higa showed that it could take up bacteriophage λ after treatment with calcium chloride solution (CaCl2).[14] Two years later, Stanley Cohen showed that CaCl2 treatment was also effective for uptake of plasmid DNA.[15] Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range.[16] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid.[17] By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants.[18]

Early genetically modified organisms[edit]

In 1972 Paul Berg utilised restriction enzymes and DNA ligases to create the first recombinant DNA molecules. He combined DNA from the monkey virus SV40 with that of the lambda virus.[19] Herbert Boyer and Stanley N. Cohen took Bergs work a step further and introduced recombinant DNA into an bacterial cell. Cohen was researching plasmids, while Boyers work involved restriction enzymes. They recognised the complimentary nature of their work and teamed up in 1972. Together they found a restriction enzyme that cut the pSC101 plasmid at a single point and were able to insert and ligate a gene that conferred resistance to the kanamycin antibiotic into the gap. Cohen had previously devised a method where bacteria could be induced to take up a plasmid and using this they were able to create a bacteria that survived in the presence of the kanamycin. This represented the first genetically modified organism. They repeated experiments showing that other genes could be expressed in bacteria, including one from the toad Xenopus laevis, the first cross kingdom transformation.[20][21][22]

In 1973 Rudolf Jaenisch created the first GM animal.

In 1973 Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.[23] Jaenisch was studying mammalian cells infected with simian virus 40 (SV40) when he happened to read a paper from Beatrice Mintz describing the generation of chimera mice. He took his SV40 samples to Mintz's lab and injected them into early mouse embryos expecting tumours to develop. The mice appeared normal, but after using radioactive probes he discovered that the virus had integrated itself into the mice genome.[24] However the mice did not pass the transgene to their offspring. In 1981 the laboratories of Frank Ruddle, Frank Constantini and Elizabeth Lacy injected purified DNA into a single-cell mouse embryo and showed transmission of the genetic material to subsequent generations.[25][26]

The first genetically engineered plant was tobacco, reported in 1983.[27] It was developed by Michael W. Bevan, Richard B. Flavell and Mary-Dell Chilton by creating a chimeric gene that joined an antibiotic resistant gene to the T1 plasmid from Agrobacterium. The tobacco was infected with Agrobacterium transformed with this plasmid resulting in the chimeric gene being inserted into the plant. Through tissue culture techniques a single tobacco cell was selected that contained the gene and a new plant grown from it.[28]

Recognition of originators[edit]

On June 19, 2013 the leaders of three research teams who originated the technology, Robert T. Fraley of Monsanto; Marc Van Montagu of Ghent University in Belgium and founder of Plant Genetic Systems and CropDesign; and Mary-Dell Chilton of Washington University in St. Louis and Syngenta were awarded with the World Food Prize. The prize, of $250,000, is awarded to people who improve the “quality, quantity or availability” of food in the world. The three competing teams first presented their results in January 1983.[29]

Regulation[edit]

The development of genetic engineering technology led to concerns in the scientific community about potential risks. The development of a regulatory framework concerning genetic engineering began in 1975, at Asilomar, California. The Asilomar meeting recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology.[30] The Asilomar recommendations were voluntary, but in 1976 the US National Institute of Health (NIH) formed a recombinant DNA advisory committee.[31] This was followed by other regulatory offices (the United States Department of Agriculture (USDA), Environmental Protection Agency (EPA) and Food and Drug Administration (FDA)), effectively making all recombinant DNA research tightly regulated in the USA.[32] In 1982 the Organization for Economic Co-operation and Development (OECD) released a report into the potential hazards of releasing genetically modified organisms into the environment as the first transgenic plants were being developed.[33] As the technology improved and genetically organisms moved from model organisms to potential commercial products the USA established a committee at the Office of Science and Technology (OSTP) to develop mechanisms to regulate the developing technology.[32] In 1986 the OSTP assigned regulatory approval of genetically modified plants in the US to the USDA, FDA and EPA.[34] In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants and food emerged from organizations including the FAO and WHO.[35][36][37][38]

Advancements[edit]

Genetically modified mice were created in 1984 that carried cloned oncogenes, predisposing them to the development of cancer.[39] The technology has also been used to generate mice with genes knocked out. The first recorded knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989. They are used to study gene function and make useful models of human diseases.[40] In 1992 oncomice with tumor suppressor genes knocked out were generated.[39] Creating Knockout rats are much harder and has only been possible since 2003.[41][42]

As not all plant cells were susceptible to infection by A. tumefaciens other methods were developed, including electroporation, micro-injection[43] and particle bombardment with the a gene gun (invented in 1987).[44][45]

In order for the gene to be incorporated into each cell the plant must undergo tissue culture. Tissue culture systems have to be developed for each plant species, with some being more susceptible than others. Methods have been developed where using vacuum infiltration (1993) or simply dipping (2008) Arabidopsis thaliana flowers in an Agrobacterium solution can result in transgenic seeds.[46] In the 1980s techniques were developed to introduce isolated chloroplasts back into a plant cell that had its cell wall removed. With the introduction of the gene gun in 1987 it became possible to integrate foreign genes into a chloroplast.[47]

Genetic engineering has been used to produce proteins derived from humans and other sources in organisms that normally cannot synthesise these proteins. Bacteria synthesising human insulin were developed in 1979, being used as a treatment for the first time in 1982.[48] In 1988 the first human antibodies were produced in plants.[49] In 1997 avidin, an egg protein, was expressed in a plant with the intention of extracting, purifying and selling it.[49] The first transgenic livestock were produced in 1985,[50] by micro injecting foreign DNA into rabbits, sheep and pigs eggs.[51] The first animal to synthesise transgenic proteins in their milk were mice,[52] engineered to produce human tissue plasminogen activator.[53] This technology has now been applied to other sheep, pigs, cows and other livestock.[52]

With the discovery of microRNA in 1993[54] came the possibility of using RNA interference to silence an organisms endogenous genes. Craig C. Mello and Andrew Fire discovered a silencing effect in 1998 through injection of double stranded RNA into C. elegans.[55] Using genetic engineering the microRNA can be expressed long term, permanently silencing the target genes. In 2002 stable gene silencing was induced in mammalian cells,[56] and in 2005 this was accomplished in a whole mouse.[57] In 2007 papers were released where insect and nematode genes that formed microRNA were put into plants, resulting in gene silencing of the pest when they ingested the transgenic plant.[58]

Commercialisation[edit]

In 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson and a year later and the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[59] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[60] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.[61] In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorization to perform field tests with the ice-minus strain of P. syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges.[62] In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment[63] when a strawberry field and a potato field in California were sprayed with it.[64] 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".[63]

The first field trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.[65] The People’s Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.[66] In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life.[67] In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first genetically engineered crop commercialized in Europe.[68] 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 USA.[69] By 2010, according to the annual ISAAA brief: "While 29 countries planted commercialized biotech crops in 2010, an additional 31 countries, totaling 60 have granted regulatory approvals for biotech crops for import for food and feed use and for release into the environment since 1996.... A total of 1,045 approvals have been granted for 196 events (NB: an "event" is a specific genetic modification in a specific species) for 25 crops. Thus, biotech crops are accepted for import for food and feed use and for release into the environment in 60 countries, including major food importing countries like Japan, which do not plant biotech crops. Of the 60 countries that have granted approvals for biotech crops, USA tops the list followed by Japan, Canada, Mexico, South Korea, Australia, the Philippines, New Zealand, the European Union, and Taiwan. Maize has the most events approved (65) followed by cotton (39), canola (15), potato and soybean (14 each). The event that has received regulatory approval in most countries is herbicide tolerant soybean event GTS-40-3-2 with 25 approvals (EU=27 counted as 1 approval only), followed by insect resistant maize MON810 with 23 approvals, herbicide tolerant maize NK603 with 22 approvals each, and insect resistant cotton (MON1445) with 14 approvals worldwide."[70]

Opposition[edit]

Opposition and support for the use of genetic engineering has existed since the technology was developed. After Arpad Pusztai went public with research he was conducting in 1998 the public opposition to genetically modified food increased.[71]

References[edit]

  1. ^ a b c d e Clive Root (2007). Domestication. Greenwood Publishing Groups. 
  2. ^ a b c Daniel Zohary, Maria Hopf, Ehud Weiss (2012). Domestication of Plants in the Old World: The origin and spread of plants in the old world. Oxford University Press. 
  3. ^ the history of maize cultivation in southern Mexico dates back 9000 years. New York Times, accessdate=2010-5-4
  4. ^ Sue Colledge and James Conolly (2007). The Origins and Spread of Domestic Plants in Southwest Asia and Europe. p. 40. 
  5. ^ a b c d e Noel Kingsbury. Hybrid: The History and Science of Plant Breeding University of Chicago Press, Oct 15, 2009
  6. ^ "Evolution of Wheatpublisher=Wheat, the big picture". 
  7. ^ Schouten, H. J.; Jacobsen, E. (2007). "Are Mutations in Genetically Modified Plants Dangerous?". Journal of Biomedicine and Biotechnology 2007: 1. doi:10.1155/2007/82612. 
  8. ^ D. L. Hartl and V. Orel (1992). "What Did Gregor Mendel Think He Discovered?". Genetics 131 (2): 245–25. 
  9. ^ Vries, H. de (1889) Intracellular Pangenesis [1] ("pan-gene" definition on page 7 and 40 of this 1910 translation in English)
  10. ^ Online copy of William Bateson's letter to Adam Sedgwick
  11. ^ Roberts, R. J. (2005). "Classic Perspective: How restriction enzymes became the workhorses of molecular biology". Proceedings of the National Academy of Sciences 102 (17): 5905–5908. doi:10.1073/pnas.0500923102. PMC 1087929. PMID 15840723. 
  12. ^ Weiss, B.; Richardson, C. C. (1967). "Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage". Proceedings of the National Academy of Sciences 57 (4): 1021. doi:10.1073/pnas.57.4.1021. 
  13. ^ Lederberg, J (1952). "Cell genetics and hereditary symbiosis". Physiological reviews 32 (4): 403–30. PMID 13003535. 
  14. ^ Mandel, Morton; Higa, Akiko (1970). "Calcium-dependent bacteriophage DNA infection". Journal of Molecular Biology 53 (1): 159–162. doi:10.1016/0022-2836(70)90051-3. PMID 4922220. 
  15. ^ Cohen, S. N.; Chang, A. C. Y.; Hsu, L. (1972). "Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA". Proceedings of the National Academy of Sciences 69 (8): 2110. doi:10.1073/pnas.69.8.2110. 
  16. ^ Wirth, Reinhard; Friesenegger, Anita and Fiedlerand, Stefan (1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation". Molecular and General Genetics MGG. 
  17. ^ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years Later)". Retrieved 14 January 2011. 
  18. ^ Zambryski, P.; Joos, H.; Genetello, C.; Leemans, J.; Montagu, M. V.; Schell, J. (1983). "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity". The EMBO Journal 2 (12): 2143–2150. PMC 555426. PMID 16453482. 
  19. ^ Jackson, D. A.; Symons, R. H.; Berg, P. (1972). "Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli". Proceedings of the National Academy of Sciences 69 (10): 2904. doi:10.1073/pnas.69.10.2904. 
  20. ^ "Genome and genetics timeline - 1973". Genome news network. 
  21. ^ Arnold, Paul (2009). "History of Genetics: Genetic Engineering Timeline". 
  22. ^ Stanley N. Cohen and Annie C. Y. Chang (1 May 1973). "Recircularization and Autonomous Replication of a Sheared R-Factor DNA Segment in Escherichia coli Transformants — PNAS". Pnas.org. Retrieved 17 July 2010. 
  23. ^ Jaenisch, R. and Mintz, B. (1974 ) Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocysts injected with viral DNA. Proc. Natl. Acad. 71(4):1250–1254 [2]
  24. ^ Brownlee, C. (2004). "Inaugural Article: Biography of Rudolf Jaenisch". Proceedings of the National Academy of Sciences 101 (39): 13982–13984. doi:10.1073/pnas.0406416101. PMC 521108. PMID 15383657. 
  25. ^ Gordon, J.; Ruddle, F. (1981). "Integration and stable germ line transmission of genes injected into mouse pronuclei". Science 214 (4526): 1244. Bibcode:1981Sci...214.1244G. doi:10.1126/science.6272397. PMID 6272397. 
  26. ^ Costantini, F.; Lacy, E. (1981). "Introduction of a rabbit β-globin gene into the mouse germ line". Nature 294 (5836): 92. Bibcode:1981Natur.294...92C. doi:10.1038/294092a0. PMID 6945481. 
  27. ^ Lemaux, P. (2008). "Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I)". Annual review of plant biology 59: 771–812. doi:10.1146/annurev.arplant.58.032806.103840. PMID 18284373. 
  28. ^ Bevan, M. W.; Flavell, R. B.; Chilton, M. D. (1983). "A chimaeric antibiotic resistance gene as a selectable marker for plant cell transformation". Nature 304 (5922): 184. doi:10.1038/304184a0. 
  29. ^ Andrew Pollack (June 19, 2013). "Executive at Monsanto Wins Global Food Honor". The New York Times. Retrieved June 20, 2013. 
  30. ^ Berg, P.; Baltimore, D.; Brenner, S.; Roblin, R. O.; Singer, M. F. (1975). "Summary statement of the Asilomar conference on recombinant DNA molecules". Proceedings of the National Academy of Sciences 72 (6): 1981. doi:10.1073/pnas.72.6.1981. 
  31. ^ Hutt, P. B. (1978). "Research on recombinant DNA molecules: The regulatory issues". Southern California law review 51 (6): 1435–50. PMID 11661661. 
  32. ^ a b McHughen A, Smyth S (2008). "US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars". Plant biotechnology journal 6 (1): 2–12. doi:10.1111/j.1467-7652.2007.00300.x. PMID 17956539.  edit
  33. ^ Bull, A.T., Holt, G. and Lilly, M.D. (1982). Biotechnology : international trends and perspectives. Paris: Organisation for Economic Co-operation and Development. 
  34. ^ U.S. Office of Science and Technology Policy (1986). "Coordinated framework for regulation of biotechnology; announcement of policy; notice for public comment". Federal register 51 (123): 23302–50. PMID 11655807. 
  35. ^ WHO (1987): Principles for the Safety Assessment of Food Additives and Contaminants in Food, Environmental Health Criteria 70. World Health Organization, Geneva
  36. ^ WHO (1991): Strategies for assessing the safety of foods produced by biotechnology, Report of a Joint FAO/WHO Consultation. World Health Organization, Geneva
  37. ^ WHO (1993): Health aspects of marker genes in genetically modified plants, Report of a WHO Workshop. World Health Organization, Geneva
  38. ^ WHO (1995): Application of the principle of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology, Report of a WHO Workshop. World Health Organization, Geneva
  39. ^ a b Hanahan, D.; Wagner, E. F.; Palmiter, R. D. (2007). "The origins of oncomice: A history of the first transgenic mice genetically engineered to develop cancer". Genes & Development 21 (18): 2258–2270. doi:10.1101/gad.1583307. PMID 17875663. 
  40. ^ "Knockout Mice". National Human Genome Research Institute. 
  41. ^ Helen R. Pilcher (2003). "It's a knockout: First rat to have key genes altered". Nature. doi:10.1038/news030512-17 (inactive 2014-04-09). 
  42. ^ Zan, Y; Haag, J. D.; Chen, K. S.; Shepel, L. A.; Wigington, D; Wang, Y. R.; Hu, R; Lopez-Guajardo, C. C.; Brose, H. L.; Porter, K. I.; Leonard, R. A.; Hitt, A. A.; Schommer, S. L.; Elegbede, A. F.; Gould, M. N. (2003). "Production of knockout rats using ENU mutagenesis and a yeast-based screening assay". Nature Biotechnology 21 (6): 645–51. doi:10.1038/nbt830. PMID 12754522. 
  43. ^ Peters, Pamela. "Transforming Plants - Basic Genetic Engineering Techniques". Retrieved 28 January 2010. 
  44. ^ Voiland, Michael; McCandless, Linda. "Development Of The "Gene Gun" At Cornell". Retrieved January 19, 2013. 
  45. ^ Roger Segelken for the Cornell Chronicle. Mary 14, 1987. Biologists Invent Gun for Shooting Cells with DNA Issue available as pdf download here, page 3
  46. ^ Clough, S. J.; Bent, A. F. (1998). "Floral dip: A simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana". The Plant Journal 16 (6): 735–743. doi:10.1046/j.1365-313x.1998.00343.x. PMID 10069079. 
  47. ^ http://www.lifesciencesfoundation.org/events-item-799.html
  48. ^ Ladisch, M. R.; Kohlmann, K. L. (1992). "Recombinant human insulin". Biotechnology Progress 8 (6): 469–478. doi:10.1021/bp00018a001. PMID 1369033. 
  49. ^ a b Woodard, S. L.; Woodard, J. A.; Howard, M. E. (2004). "Plant molecular farming: Systems and products". Plant Cell Reports 22 (10): 711–720. doi:10.1007/s00299-004-0767-1. PMID 14997337.  edit
  50. ^ Brophy, B.; Smolenski, G.; Wheeler, T.; Wells, D.; l'Huillier, P.; Laible, G. T. (2003). "Cloned transgenic cattle produce milk with higher levels of β-casein and κ-casein". Nature Biotechnology 21 (2): 157–162. doi:10.1038/nbt783. PMID 12548290. 
  51. ^ Hammer, R. E.; Pursel, V. G.; Rexroad, C. E.; Wall, R. J.; Bolt, D. J.; Ebert, K. M.; Palmiter, R. D.; Brinster, R. L. (1985). "Production of transgenic rabbits, sheep and pigs by microinjection". Nature 315 (6021): 680–683. doi:10.1038/315680a0. PMID 3892305. 
  52. ^ a b A. John Clark. "The Mammary Gland as a Bioreactor: Expression, Processing, and Production of Recombinant Proteins". Journal of Mammary Gland Biology and Neoplasia 3 (3): 337–350. doi:10.1023/a:1018723712996. 
  53. ^ K. Gordon, E. Lee, J. Vitale, A. Smith, H. Westphal, and L. Hennighausen (1987). "Production of human tissue plasmnogen activator in transgenic mouse milk". Biotechnology 5: 1183±1187. doi:10.1038/nbt1187-1183. 
  54. ^ Lee, R.C.; Ambros, V. (1993). "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.". Cell. 
  55. ^ Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391 (6669): 806–811. doi:10.1038/35888. PMID 9486653.  edit
  56. ^ Brummelkamp, T. R.; Bernards, R.; Agami, R. (2002). "A System for Stable Expression of Short Interfering RNAs in Mammalian Cells". Science 296 (5567): 550–553. doi:10.1126/science.1068999. PMID 11910072.  edit
  57. ^ Peng, S. (2006). "A transgenic approach for RNA interference-based genetic screening in mice". Proceedings of the National Academy of Sciences 103 (7): 2252–2220. doi:10.1073/pnas.0511034103. 
  58. ^ Vaucheret, H.; Chupeau, Y. (2011). "Ingested plant miRNAs regulate gene expression in animals". Cell Research 22 (1): 3–5. doi:10.1038/cr.2011.164. PMC 3351922. PMID 22025251. 
  59. ^ Goeddel, D. V.; Kleid, D. G.; Bolivar, F.; Heyneker, H. L.; Yansura, D. G.; Crea, R.; Hirose, T.; Kraszewski, A.; Itakura, K.; Riggs, A. D. (1979). "Expression in Escherichia coli of chemically synthesized genes for human insulin". Proceedings of the National Academy of Sciences 76: 106. doi:10.1073/pnas.76.1.106. 
  60. ^ US Supreme Court Cases from Justia & Oyez (16 June 1980). Diamond V Chakrabarty 447. Supreme.justia.com. Retrieved 17 July 2010. 
  61. ^ "Artificial Genes". TIME. 15 November 1982. Retrieved 17 July 2010. 
  62. ^ Rebecca Bratspies (2007) Some Thoughts on the American Approach to Regulating Genetically Modified Organisms. Kansas Journal of Law and Public Policy 16:393 [3]
  63. ^ a b BBC News 14 June 2002 GM crops: A bitter harvest?
  64. ^ Thomas H. Maugh II for the Los Angeles Times. June 09, 1987. Altered Bacterium Does Its Job : Frost Failed to Damage Sprayed Test Crop, Company Says
  65. ^ James, Clive (1996). "Global Review of the Field Testing and Commercialization of Transgenic Plants: 1986 to 1995". The International Service for the Acquisition of Agri-biotech Applications. Retrieved 17 July 2010. 
  66. ^ James, Clive (1997). "Global Status of Transgenic Crops in 1997". ISAAA Briefs No. 5.: 31. 
  67. ^ Bruening, G.; Lyons, J. M. (2000). "The case of the FLAVR SAVR tomato". California Agriculture 54 (4): 6–7. doi:10.3733/ca.v054n04p6.  edit
  68. ^ Debora MacKenzie (18 June 1994). Transgenic tobacco is European first. New Scientist. 
  69. ^ Genetically Altered Potato Ok'd For Crops Lawrence Journal-World - 6 May 1995
  70. ^ Global Status of Commercialized Biotech/GM Crops: 2011 ISAAA Brief ISAAA Brief 43-2011. Retrieved 14 October 2012
  71. ^ Arpad Pusztai: Biological divide James Randerson The Guardian January 15, 2008