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Transgenesis

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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.

Transgenic organisms are able to express foreign genes 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 protein sequence, scientists can cut DNA at these common protein points and add other genes. An example of this is the "super mice" of the 1980s. These mice were able to produce the human protein tPA to treat blood clots.

Using plasmids from bacteria

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 Plasmid for replication.[2]

Gene Transfer Technology

DNA Microinjection

The Desired gene construct is injected in the pronucleus of a reproductive cell using a glass needle around 0.5 to 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 microinjection 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 new traits will not appear in their offspring. If DNA is injected in multiple sites the chances of over-expression increase.[3]

Retrovirus-mediated gene Transfer

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 inbred for as many as 20 generations until homozygous genetic offspring are born.[3]

Stem Cell Transgenesis

Pluripotent Stem Cell Transgenesis

Transgenic vectors can be delivered randomly[citation needed], or targeted to a specific genomic location, such as a safe harbor [citation needed]. A broad range of scientists from the transgenesis community have already performed a remarkable amount of impressive research and technology development to provide the tools necessary to permit safe and effective pluripotent stem cell (PSC) transgenesis.[4][5][6][7][8][9][10][11] This transgenic PSC technology could be applicable to endogenous gene correction, gene replacement therapy, or the addition of a transgenic safety element in hiPSCs or to a combination of these. Two of the major safety concerns regarding hiPSC-based therapeutics are (1) the residual presence of PSCs in the transplanted stem cell graft and (2) the formation of malignant cells from the stem cell derivatives in the short, medium, or long term.[12] There are a large number of recent studies demonstrating that safe-harbor based transgenic technology may be able to solve many of the current clinical roadblocks in the stem cell therapy field, if the PSC transgenesis process incorporates novel technologies, such as CRISPR/Cas9-nickase based complex transgenic multiplexing[citation needed] and other more advanced forms of stem cellgenetic engineering.

Totipotent Stem Cell Transgenesis

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 into the host’s embryo, resulting in a chimeric animal. Unlike the other two methods of injection which require live transgenic offspring for testing, embryonic cell transfer can be tested at the cell stage.

Pharming

Main article: Pharming (genetics)

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
  • alpha-1 antitrypsin (AAT) for treatment of AAT deficiency
  • insulin for diabetes treatment
  • 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

Diagram

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

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Note: New genotypes created with transgenic technologies also require multiple backcrossings. Furthermore, backcrossing does not account for the majority of time required to create, field test and release/commercialize a new variety.

References

  1. ^ "FAS - Mousepox Case Study - Module 4.0". Federation of American Scientists. History of Transgenics. Archived from the original on July 15, 2007. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  2. ^ a b Redway, Keith. "Transgenic organisms". Gene Manipulation & Recombinant DNA. University of Westminster. Retrieved June 28, 2014. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  3. ^ a b Margawati, Endang Tri (January 2003). "Transgenic Animals: Their Benefits To Human Welfare". Actionbioscience. Retrieved June 29, 2014. {{cite web}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  4. ^ Capecchi, M.R., Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet., 2005. 6(6): p. 507-12.
  5. ^ Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23.
  6. ^ DiCarlo, J.E., et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res., 2013. 41(7): p. 4336-43.
  7. ^ Friedland, A.E., et al., Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 2013. 10(8): p. 741-3.
  8. ^ Hwang, W.Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013. 31(3): p. 227-9.
  9. ^ Nguyen, H.N. and R. Reijo Pera, Metaphase spreads and spectral karyotyping of human embryonic stem cells. CSH Protoc., 2008. 2008: p. pdb.prot5047.
  10. ^ Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6.
  11. ^ Xue, H., et al., Genetic modification in human pluripotent stem cells by homologous recombination and CRISPR/Cas9 system. Methods Mol Biol., 2014. Mar 11. [Epub ahead of print].
  12. ^ Chung, S., et al., Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation. J Neurochem., 2006. 97(5): p. 1467-80.
  13. ^ Lopatto, Elizabeth (October 1, 2012). "Gene-Modified Cow Makes Milk Rich in Protein, Study Finds". Bloomberg Businessweek. New York City. {{cite news}}: Unknown parameter |deadurl= ignored (|url-status= suggested) (help)
  14. ^ http://people.ucalgary.ca/~browder/transgenic.html
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