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Stem cell transgenesis

By using a stem cell as the vehicle to introduce the exogenous gene, the stem cell can conform to the cell type around it, expressing the newly introduced gene. Embryonic cells (or cells that have been coaxed back into a stem cell state) are modified and selected for a particular trait or gene, and then injected into a developing blastocyst in vivo, resulting in modified offspring^[1]. Experiments using embryonic stem cells have shown that 10% of the injected blastocysts express the chimeric genes^[1] onced carried to term, a similar effectiveness to microinjection and retrovirus methods. There are some benefits to using stem cells as the vehicle, such as the ability to completely screen the condition and traits of the injected cell, so scientists can better keep track of the traits that are being introduced.

Stem cell transgenesis can be divided into three parts, named for the utilized stem cells' ability to differentiate: Multipotent, Pluripotent, and Totipotent.

Multipotent stem cell transgenesis

Multipotent stem cells can only differentiate into a limited number of therapeutically useful cell types, nevertheless their safety and relative lack of complexity have resulted in the vast majority of current personalized stem-cell therapy involving multipotent stem cells (typically mesenchymal stem cells from adipose tissue).^[2]

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]}. Scientists have performed research and technology development to provide the tools necessary to permit safe and effective pluripotent stem cell (PSC) transgenesis.^[3]^[4]^[5]^[6]^[7]^[8]^[9]^[10]

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.

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^ ^a ^b Gossler, Achim (15 August 1986). "Transgenesis by means of blastocyst derived embryonic stem cell lines" (PDF). Proceedings of the National Academy of Sciences.
^ "Clinical Trials | MD Anderson Cancer Center". clinicaltrials.org. Retrieved 2016-02-24.
^ 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.
^ Cong L, Ran FA, Cox D, et al. (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–23. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.
^ 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ Hwang WY, Fu Y, Reyon D, et al. (March 2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nat. Biotechnol. 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.
^ Nguyen HN, Reijo Pera RA (2008). "Metaphase spreads and spectral karyotyping of human embryonic stem cells". CSH Protoc: pdb.prot5047. PMID 21356916.
^ 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.
^ 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)

[:0-1] Gossler, Achim (15 August 1986). "Transgenesis by means of blastocyst derived embryonic stem cell lines" (PDF). Proceedings of the National Academy of Sciences.

[2] "Clinical Trials | MD Anderson Cancer Center". clinicaltrials.org. Retrieved 2016-02-24.

[3] 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.

[4] Cong L, Ran FA, Cox D, et al. (February 2013). "Multiplex genome engineering using CRISPR/Cas systems". Science. 339 (6121): 819–23. doi:10.1126/science.1231143. PMC 3795411. PMID 23287718.

[5] 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)

[6] 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)

[7] Hwang WY, Fu Y, Reyon D, et al. (March 2013). "Efficient genome editing in zebrafish using a CRISPR-Cas system". Nat. Biotechnol. 31 (3): 227–9. doi:10.1038/nbt.2501. PMC 3686313. PMID 23360964.

[8] Nguyen HN, Reijo Pera RA (2008). "Metaphase spreads and spectral karyotyping of human embryonic stem cells". CSH Protoc: pdb.prot5047. PMID 21356916.

[9] 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.

[10] 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)

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