Abstract
For many years the lack of germline competent embryonic stem cell lines in livestock meant that the targeted modification of endogenous genes was not possible in these species. The demonstration that livestock could be cloned by nuclear transfer from cultured somatic cells has now provided an alternative route to accomplish gene targeting. This chapter describes protocols for culturing primary sheep fibroblasts, introducing and selecting targeted modifications into them and then using these modified cells in nuclear transfer experiments.
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References
Hammer, R. E., Pursel, V. G., Rexroad, C., et al. (1985) Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315, 680–683.
Clark, A. J., Bissinger, P., Bullock, D. W., et al. (1994) Chromosomal position effects and the modulation of transgene expression. Reprod Fertil Dev. 6, 589–598.
Hooper, M. L. (1992) Embryonal Stem Cells: Introducing Planned Changes Into the Germline (Evans, H. J., ed). Harwood Academic Publishers, Switzerland.
Stice, S. L. (1998) Opportuities and challenges in domestic animal embryonic stem cell research, in Animal Breeding: Technology for the 21st Century (Clark, A. J., ed.) Harwood Aacademic Press, Switzerland, pp. 64–71.
Wheeler, M. (1994). Development and validation of swine embryonic stem cells: a review. Reprod. Fertil. Dev. 6, 563–568.
Campbell, K. H.S., McWhir, J., Ritchie, W. A., and Wilmut, I. (1996). Sheep cloned by nuclear transfer from a cultured cell line. Nature 380, 64–66.
Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H.S. (1997). Viable offspring derived from fetal and adult mammalian cells Nature 385, 810–813.
Te Riele, H., Robanus, M., and Berns, A. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs Proc. Natl. Acad. Sci. USA 89, 5128–5132.
Deng, C. and Capecchi, M. R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell Biol. 12, 3365–3371.
Shulman, M. J., Nissen, L., and Collins, C. (1990) Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol. Cell Biol. 10, 4466–4472.
Thomas, K. R. and Capecchi, M. R. (1990) Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346, 847–580.
Thomas, K. R., Deng, C., and Capecchi, M. R. (1992) High-fidelity gene targeting in embryonic stem cells by using sequence replacement vectors. Mol. Cell Biol. 12, 2919–2923.
Sedivy, J. M. and Sharp, P. A. (1989) Positive genetic selection for gene disruption in mammalian cells by homologous recombination. Proc. Natl. Acad. Sci. USA 86, 227–231.
Sedivy, J. (1999) Gene targeting in human cells without isogenic DNA. Science 283, 9a.
Jeannotte, L., Ruiz, J. C., and Robertson, E. J. (1991) Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption. off. Mol. Cell Biol. 11, 5578–5585.
Shichiri, M., Hanson, K. D., and Sedivy, J. M. (1993) Effects of c-myc expression on proliferation, quiescence, and the G0 to G1 transition in nontransformed cells. Cell Growth Differ. 4, 93–104.
Mansour, S. L., Thomas, K. R., and Capecchi, M. R. (1988) Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature 336, 348–352.
Jasin, M. and Berg, P. (1988) Homologous integration in mammalian cells without target gene selection. Genes Dev. 2, 1353–1363.
Yanagawa, Y., Kobayashi, T., Ohnishi, M., et al. (1999) Enrichment and efficient screening of ES cells containing a targeted mutation: the use of DT-A gene with the polyadenylation signal as a negative selection maker. Transgenic Res. 8, 215–221.
Hanson, K. D. and Sedivy, J. M. (1995) Analysis of biological selections for high-efficiency gene targeting. Mol. Cell Biol. 15, 45–51.
Denning C, Burl S, Ainslie A, et al. (2001) Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep. Nat. Biotechnol. 19 559–562.
Clark, A. J., Burl, S., Denning, C., and Dickinson, P. (2000). Gene targeting in livestock; a preview. Transgenic Res. 9, 263–275.
Clark, A. J., Ferrier, P., Aslam, S., et al. (2003) Proliferative lifespan is conserved after nuclear transfer. Nat Cell Biol. 5, 535–538.
Yanez, R. J. and Porter, A. C.G. (1998) Therapeutic gene targeting. Gene Ther. 5, 149–159.
Denning, C., Dickinson, P., Burl, S., Wylie, D., Fletcher, J., and Clark, A. J. (2001) Gene targeting in primary fetal fibroblasts from sheep and pig. Cloning Stem Cells. 3, 221–231.
McCreath, K. J., Howcroft, J., Campbell, K. H., Colman, A., Schnieke, A. E., and Kind, A. J. (2000). Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature. 405, 1066–1069.
Cui, W., Aslam, S., Fletcher, J., Wylie, D., Clinton. M., and Clark, A. J. (2002) Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts. J. Biol. Chem. 277, 38,531–38,539.
Cui, W., Wylie, D., Aslam, S., et al. (2003) Telomerase-immortalized sheep fibroblasts can be reprogrammed by nuclear transfer to undergo early development. Biol. Reprod. 69, 15–21.
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Clark, A.J., Burl, S., Denning, C. (2006). Genetic Modification of Sheep by Nuclear Transfer With Gene-Targeted Somatic Cells. In: Verma, P.J., Trounson, A.O. (eds) Nuclear Transfer Protocols. Methods in Molecular Biology™, vol 348. Humana Press. https://doi.org/10.1007/978-1-59745-154-3_13
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DOI: https://doi.org/10.1007/978-1-59745-154-3_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-280-3
Online ISBN: 978-1-59745-154-3
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