Transgenesis

Transgenesis 2.2 Retrovirus-mediated gene transfer

Transgenesis is the process of introducing an exogenous gene — called a transgene — into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. Transgenesis can be facilitated by liposomes, plasmid vectors, viral vectors, pronuclear injection, protoplast fusion, and ballistic DNA injection.

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA. Retroviruses are used as vectors to transfer genetic material into the host cell. The result is a chimera, an organism consisting of tissues or parts of diverse genetic constitution. Chimeras are inuntil homozygous geTransgenic organisms are able to express foreign genes bred for as many as 20 generations [3] netic offspring are born. because the genetic code is similar for all organisms. This means that a specific DNA sequence will code for the same protein in all organisms. Due to this similarity in 2.3 Stem cell transgenesis protein sequence, scientists can cut DNA at these common protein points and add other genes. An example of 2.3.1 Multipotent stem cell transgenesis this is the “super mice” of the 1980s. These mice were able to produce the human protein tPA to treat blood Multipotent stem cells can only differentiate into a limclots. ited number of therapeutically useful cell types, nevertheless their safety and relative lack of complexity to us have resulted in the vast majority of current personalized cellular therapeutics involving multipotent stem cells (typi1 Using plasmids from bacteria cally mesenchymal stem cells from adipose tissue).[4] The most common type of transgenesis research is done with bacteria and viruses which are able to replicate foreign DNA.[1] The plasmid DNA is cut using restriction enzymes, while the DNA to be copied is also cut with the same restriction enzyme, producing complementary sticky-ends. This allows the foreign DNA to hybridise with the plasmid DNA and be sealed by DNA ligase enzyme, creating a genetic code not normally found in nature. Altered DNA is inserted into plasmids for replication.[2]

2.3.2 Pluripotent stem cell transgenesis Transgenic vectors can be delivered randomly, or targeted to a specific genomic location, such as a safe harbor . Scientists have performed research and technology development to provide the tools necessary to permit safe and effective pluripotent stem cell (PSC) transgenesis.[5][6][7][8][9][10][11][12] 2.3.3 Totipotent stem cell transgenesis

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Gene transfer technology

The manipulated gene construct is inserted into totipotent stem cells, cells which can develop into any specialized cell. Cells containing the desired DNA are incorporated 2.1 DNA microinjection into the host’s embryo, resulting in a chimeric animal. Unlike the other two methods of injection which require The Desired gene construct is injected in the pronucleus live transgenic offspring for testing, embryonic cell transof a reproductive cell using a glass needle around 0.5 to fer can be tested at the cell stage. 5 micrometers in diameter. The manipulated cell is cultured in vitro to develop to a specific embryonic phase, is then transferred to a recipient female. DNA microinjec3 Applications tion does not have a high success rate (roughly 2% of all injected subjects), even if the new DNA is incorporated in the genome, if it is not accepted by the germ-line the 3.1 Pharming new traits will not appear in their offspring. If DNA is injected in multiple sites the chances of over-expression Main article: Pharming (genetics) increase.[3] 1

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Pharming is a portmanteau of “farming” and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes, thus creating a genetically modified organism (GMO). Pharming has gained application in biotechnology since the development of trangsgenic “super mice” in 1982. “Super mice” were genetically altered to produce the human drug, tPA (tissue plasminogen activator to treat blood clots), in 1987.[2] Since then “super mice” pharming has come a long way. Using RNA interference scientists have produced a cow whose milk contains increased amounts of casein, a protein used to make cheese and other foods, and almost no beta-lactoglobulin, a component in milk whey protein that causes allergies.[13] “Pharming Examples:"[14] • Haemoglobin as a blood substitute • human protein C anticoagulant

Conventional breeding Current cultivar

Transgenesis

Wild relative

Cisgenesis Wild relative

Unrelated organism

Beneficial gene

Beneficial gene

Unwanted genes

Beneficial gene

Unwanted genes

Current cultivar

Back cross

REFERENCES

F1 Generation

Transfer into plasmid in Agrobacterium

Unwanted genes

Current cultivar

Transfer into plasmid in Agrobacterium Genes required for transfer

Genes required for transfer

F2 Generation

Many backcrosses

New cultivar

New cultivar

New cultivar

A diagram comparing the genetic changes achieved through conventional plant breeding, transgenesis and cisgenesis

5 Diagram .

• alpha-1 antitrypsin (AAT) for treatment of AAT de- Note: New genotypes created with transgenic technologies also require multiple backcrossings. Furthermore, ficiency backcrossing does not account for the majority of time required to create, field test and release/commercialize a • insulin for diabetes treatment new variety. • vaccines (antigens) • growth hormones for treatment of deficiencies • factor VIII blood clotting factor • factor IX blood clotting factor • fibrinogen blood clotting factor • lactoferrin as an infant formula additive

3.2

Medical

Transgenesis can be used to neutralize genes that would normally prevent xenotransplantation. For example, a protein found in pigs can cause humans to reject their transplanted organs. This protein can be replaced by a similar human genome to prevent the rejection.[15]

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Ethical Concerns

Transgenesis has created certain ethical concerns. Examples include rights for animals that have been improved intellectually, legal ramifications, and possible health risks.[16]

6 References [1] “Mousepox Case Study — Module 4.0”. Federation of American Scientists. History of Transgenics. Archived from the original on July 15, 2007. [2] Redway, Keith. “Transgenic organisms”. Gene Manipulation & Recombinant DNA. University of Westminster. Retrieved June 28, 2014. [3] Margawati, Endang Tri (January 2003). “Transgenic Animals: Their Benefits To Human Welfare”. Actionbioscience. Retrieved June 29, 2014. [4] clinicaltrials.org [5] Capecchi MR (June 2005). “Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century”. Nat. Rev. Genet. 6 (6): 507–12. doi:10.1038/nrg1619. PMID 15931173. [6] Cong L, Ran FA, Cox D, et al. (February “Multiplex genome engineering using 2013). CRISPR/Cas systems”. Science 339 (6121): 819– 23. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718. [7] DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (April 2013). “Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems”. Nucleic Acids Res. 41 (7): 4336–43. doi:10.1093/nar/gkt135. PMC 3627607. PMID 23460208.

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[8] Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA (August 2013). “Heritable genome editing in C. elegans via a CRISPR-Cas9 system”. Nat. Methods 10 (8): 741–3. doi:10.1038/nmeth.2532. PMC 3822328. PMID 23817069. [9] Hwang WY, Fu Y, Reyon D, et al. (March 2013). “Efficient genome editing in zebrafish using a CRISPRCas system”. Nat. Biotechnol. 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964. [10] Nguyen HN, Reijo Pera RA (2008). “Metaphase spreads and spectral karyotyping of human embryonic stem cells”. CSH Protoc: pdb.prot5047. PMID 21356916. [11] Mali P, Yang L, Esvelt KM, et al. (February 2013). “RNA-guided human genome engineering via Cas9”. Science 339 (6121): 823–6. doi:10.1126/science.1232033. PMC 3712628. PMID 23287722. [12] Xue H, Wu J, Li S, Rao MS, Liu Y (March 2014). “Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System”. Methods Mol. Biol. doi:10.1007/7651_2014_73. PMID 24615461. [13] Lopatto, Elizabeth (October 1, 2012). “Gene-Modified Cow Makes Milk Rich in Protein, Study Finds”. Bloomberg Businessweek (New York City). [14] Buy M (1997). “Transgenic Animals”. CCAC Resource Supplement. Canadian Council on Animal Care (CCAC). [15] “Actionbioscience | Transgenic Animals: Their Benefits To Human Welfare”. actionbioscience.org. Retrieved November 29, 2014. [16] “Actionbioscience | Ethical Issues in Genetic Engineering and Transgenics”. actionbioscience.org. Retrieved November 29, 2014.

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7 TEXT AND IMAGE SOURCES, CONTRIBUTORS, AND LICENSES

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Text and image sources, contributors, and licenses

7.1

Text

• Transgenesis Source: http://en.wikipedia.org/wiki/Transgenesis?oldid=639654588 Contributors: Xanzzibar, Discospinster, Rich Farmbrough, Melaen, Ceyockey, RHaworth, Nightryder84, SmackBot, RDBrown, Kikumbob, Gogo Dodo, Hebrides, JustAGal, Prolog, Smartse, Dougher, R'n'B, Idioma-bot, Birczanin, Enviroboy, MaCRoEco, Genya Avocado, Forest Ash, ClueBot, Unbuttered Parsnip, Roadfish, BOTarate, MystBot, Addbot, Thymo, LaaknorBot, F Notebook, Luckas-bot, Yobot, Fraggle81, AnomieBOT, GrouchoBot, Omnipaedista, FrescoBot, Steve Quinn, Mparu, Aircorn, ZéroBot, Joonoob, Nicepirate, ClueBot NG, Snotbot, Brickrosco, Rhall28, Willnabors, Evanbrem, BG19bot, Bonnie13J, Naapple, Webclient101, Calerc, Dmitry Dzhagarov, Hillbillyholiday, Deceiving Gods, Jim-Siduri, Jordanjlatimer and Anonymous: 28

7.2

Images

• File:Breeding_transgenesis_cisgenesis.svg Source: http://upload.wikimedia.org/wikipedia/commons/8/80/Breeding_transgenesis_ cisgenesis.svg License: ? Contributors: Transferred from en.wikipedia; transferred to Commons by User:Tm using CommonsHelper. Original artist: Smartse (talk). Original uploader was Smartse at en.wikipedia • File:Question_book-new.svg Source: http://upload.wikimedia.org/wikipedia/en/9/99/Question_book-new.svg License: Cc-by-sa-3.0 Contributors: Created from scratch in Adobe Illustrator. Based on Image:Question book.png created by User:Equazcion Original artist: Tkgd2007

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Content license

• Creative Commons Attribution-Share Alike 3.0