Challenges and Prospects for Targeted Transgenesis in Livestock

Practical Applications of Gene Targeting
  • Margarita M. Marques
  • Alison J. Thomson
  • Jim McWhir
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 534)


Until recently the only ways of making transgenic livestock were by pro-nuclear injection or, more controversially, by sperm mediated DNA transfer. Both techniques usually result in multiple copies of the transgene at a random site within the genome. This is associated with unpredictable transgene expression often due to gene silencing. In the mouse, this problem has been addressed by directing single copy transgenes to particular sites in the genome (gene targeting). This has been possible because of the availability, in that species, of embryo-derived stem cells (ES cells) which can be modifiedin vitroand then used to create transgenic mice. Despite considerable research effort, however, ES cells are not available for any livestock species. ES-like cells have been derived from sheep, pigs and cattle that can contribute to the formation of chimaeras but they do not contribute to the germline (reviewed by McWhir).


Cystic Fibrosis Homologous Recombination Telomere Length Nuclear Transfer Replicative Senescence 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bronson, S.K., Plaehn, E.G., Kluckman, K.D., Hagamann, J.R., Maeda, N., and Smithies, O., 1996, Single-copy transgenic mice with chosen-site integration.Proc. Natl. Acad. Sci. USA93, 9067–9072.CrossRefGoogle Scholar
  2. 2.
    Wallace, H., Ansell, R., Clark, J., and McWhir, J., 2000, Pre-selection of integration sites imparts repeatable transgene expression.Nucleic Acids Res.28, 1455–1464.CrossRefGoogle Scholar
  3. 3.
    Evans, M.J., and Kaufman, M.H., 1981, Establishment in culture of pluripotential cells from mouse embryos.Nature292, 154–156.CrossRefGoogle Scholar
  4. 4.
    McWhir, J., 1999, Biomedical and Agricultural Applications of Animal Transgenesis. In Transgenesis Techniques (A. R. Clarke, ed), Humana Press, Totowa, N.J.Google Scholar
  5. 5.
    Campbell, K.H., McWhir, J., Ritchie, W.A., and Wilmut, I., 1996, Sheep cloned by nuclear transfer from a cultured cell line.Nature380, 64–66.CrossRefGoogle Scholar
  6. 6.
    Dai., Y., Vaught, T.D., Boone, J., Chen, S-H., Phelps, C.J., Ball, S., Monahan, J.A., Jobst, P.M., McCreath, K.J., Lamborn, A.E., Cowell-Lucero, J.L., Wells, K.D., Colman, A., Polejaeva, I.A., and Ayares, D.L., 2002, Targeted disruption of the a-1,3galactosyltransferase gene in cloned pigs.Nature Biotechnol.20, 251–255.CrossRefGoogle Scholar
  7. 7.
    Lai, L., Kolber-Simonds, D., Park, K-W., Cheong, H-T., Greenstein, J.L., Im, G-S., Samuel, M., Bonk, A., Rieke, A., Day, B.N., Murphy, C.N., Carter, D.B., Hawley, R.J., and Prather, R.S., 2002, Production of a-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.Science295, 1089–1092.CrossRefGoogle Scholar
  8. 8.
    Devinoy, E., Thepot, D., Stinnakre, M. G., Fontaine, M. L., Grabowski, H., Puissant, C., Pavirani, A., and Houdebine, L. M., 1994, High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland.Transgenic Res.3, 79–89.CrossRefGoogle Scholar
  9. 9.
    Platenburg, G.J., Kootwijk, E.P., Kooiman, P.M., Woloshuk, S.L., Nuijens, J.H., Krimpenfort, P.J., Pieper, F.R., de Boer, H.A., and Strijker, R., 1994, Expression of human lactoferrin in milk of transgenic mice.Transgenic Res.3, 99–108.CrossRefGoogle Scholar
  10. 10.
    Massoud, M., Attal, J., Thepot, D., Pointu, H., Stinnakre, M. G., Theron, M. C., Lopez, C., and Houdebine, L. M., 1996, The deleterious effects of human erythropoietin gene driven by the rabbit whey acidic protein gene promoter in transgenic rabbits.Reprod. Nutr. Dey.36, 555–563.CrossRefGoogle Scholar
  11. 11.
    Zinovieva, N., Lassnig, C., Schams, D., Besenfelder, U., Wolf, E., Muller, S., Frenyo, L., Seregi, J., Muller, M., and Brem, G., 1998, Stable production of human insulin-like growth factor 1 (IGF-1) in the milk of hemi-and homozygous transgenic rabbits over several generations.Transgenic Res.7, 437–447.CrossRefGoogle Scholar
  12. 12.
    Wright, G., Carver, A., Cottom, D., Reeves, D., Scott, A., Simons, P., Wilmut, I., Garner, I., and Colman, A., 1991, High level expression of active human alpha-l-antitrypsin in the milk of transgenic sheep.Biotechnology (NY)9, 830–834.CrossRefGoogle Scholar
  13. 13.
    Swanson, M.E., Martin, M.J, O’Donnell, J.K., Hoover, K., Lago, W., Huntress, V., Parsons, C.T., Pinkert, C.A., Pilder, S., and Logan, J.S., 1992, Production of functional human hemoglobin in transgenic swine.Biotechnology (NY)10, 557–559.CrossRefGoogle Scholar
  14. 14.
    Kuroiwa, Y., Yoshida, H., Ohshima, T., Shinohara, T., Ohguma, A., Kazuki, Y., Oshimura, M., Ishida, I., and Tomizuka, K., 2002, The use of chromosome-based vectors for animal transgenesis.Gene Ther.9, 708–712.CrossRefGoogle Scholar
  15. 15.
    Dobie, K.W., Lee, M., Fantes, J.A., Graham, E., Clark, A.J., Springbett, A., Lathe, R., and McClenaghan, M., 1996, Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.Proc. Natl. Acad. Sci. USA93, 6659–6664.CrossRefGoogle Scholar
  16. 16.
    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.Nature405, 1066–1069.CrossRefGoogle Scholar
  17. 17.
    McClenaghan, M., Archibald, A.L., Harris, S., Simons, J.P., Whitelaw, C.B.A., Wilmut, I., and Clark, A.J., 1991, Production of Human alphal -antitrypsin in the milk of transgemic sheep and mice: Targeting expression of eDNA sequences to the mammary gland.Animal Biotechnology2, 161–176.CrossRefGoogle Scholar
  18. 18.
    Carver, A., Wright, G., Cottom, D., Cooper, J., Dalrymple, M., Temperley, S., Udell, M., Reeves, D., Percy, J., Scott, al.1992, Expression of human alpha 1 antitrypsin in transgenic sheep. Cytotechnology 9, 77–84.CrossRefGoogle Scholar
  19. 19.
    Kumar, S., Clarke, A.R., Hooper, M.L., Home, D.S., Law, A.J., Leaver, J., Springbett, A., Stevenson, E., and Simons, J.P., 1994, Milk composition and lactation of beta-caseindeficient mice.Proc. Natl. Acad. Sci. USA91, 6138–6142.CrossRefGoogle Scholar
  20. 20.
    Clark, A.J. 1996, Genetic modification of milk proteins.Am. J. Clin. Nutr63,633S–638S.Google Scholar
  21. 21.
    Müller, M., and Brem, G., 1994, Transgenic strategies to increase disease resistance in livestock.Reprod. Fertil. Del6, 605–613.CrossRefGoogle Scholar
  22. 22.
    Büeler, H., Aguzzi, A, Sailer, A., Greiner, R.-A., Autenried, P., Aguet, M., and Weissmann, C., 1993, Mice devoid of PrP are resistant to scrapie.Cell73, 1339–1347.CrossRefGoogle Scholar
  23. 23.
    Tobler, I, Gaus, S. E., Deboer, T., Achermann, P., Fischer, M., Rulicke, T., Moser, M., Oesch, B., McBride, P.A., and Manson, J.C., 1996, Altered circadian activity rhythms and sleep in mice devoid of prion protein.Nature380, 639–642.CrossRefGoogle Scholar
  24. 24.
    Nishida, N., Katamine, S., Shigematsu, K., Nakatani, A., Sakamoto, N., Hasegawa, S., Nakaoke, R, Atarashi, R., Kataoka, Y., and Miyamoto, T., 1997, Prion protein is necessary for latent learning and long-term memory retention.Cell Mol. Neurobiol.17, 537–545.CrossRefGoogle Scholar
  25. 25.
    Denning, C., Burl, S., Ainslie, A., Bracken, J., Dinnyes, A., Fletcher, J., King, T., Ritchie, M., Ritchie, W.A., Rollo, M., de Sousa, P., Travers, A., Wilmut, I., and Clark, A.J., 2001, Deletion of the alpha(1,3)galactosy1 transferase (GGTA 1) gene and the prion protein (PrP) gene in sheep.Nat. Biotechnol.19, 559–562.CrossRefGoogle Scholar
  26. 26.
    Maga, E.A., Anderson, G.B., and Murray, J.D., 1995, The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice.J. Dairy Sci.78, 2645–2652.CrossRefGoogle Scholar
  27. 27.
    Kerr, D.E., Plaut, K., Bramley, A.J., Williamson, C.M., Lax, A.J., Moore, K, Wells, K.D., and Wall, RJ., 2001, Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.Nature Biotechnol.19, 66–70.CrossRefGoogle Scholar
  28. 28.
    Snouwaert, J.N., Brigman, K.K., Latour, A.M., Malouf, N.N., Boucher, R.C., Smithies, O., and Koller, B.H., 1992, An animal model for cystic fibrosis made by gene targeting.Science257, 1083–1088.CrossRefGoogle Scholar
  29. 29.
    Harris, A., 1997, Towards an ovine model of cystic fibrosis.Hum. Mol. Genet.6, 2191–2194.CrossRefGoogle Scholar
  30. 30.
    Patience, C., Takeuchi, Y., and Weiss, R. A., 1997, Infection of human cells by an endogenous retrovirus of pigs.Nature Med.3, 282–286.CrossRefGoogle Scholar
  31. 31.
    Logan, J.S., 2000, Prospects for xenotransplantation.Curr. Opin. Inmunol.12, 563–568.CrossRefGoogle Scholar
  32. 32.
    Galili, U., 2001, The a-gal epitope (Gala1–3Galkkkkkk1–4G1cNAc-R) in xenotransplantation.Biochimie83,557–563.CrossRefGoogle Scholar
  33. 33.
    Harrison, S.J., Guidolin, A., Faast, R., Crocker, L.A., Giannakis, C., d’Apice, A.J.F., Nottle, M.B. and Lyons, I., 2002, Efficient generation of a (1,3) galactosyltransferase knockout porcine fetal fibroblasts for nuclear transfer.Transgenic Res.11, 143–150.CrossRefGoogle Scholar
  34. 34.
    Tearle, R.G., 1996, The the a-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.Transplantation61, 13–19.CrossRefGoogle Scholar
  35. 35.
    Platt, J.L., 2002, Knocking out xenograft rejection.Nature Biotechnol.20, 231–232.CrossRefGoogle Scholar
  36. 36.
    Hayflick, L., 1965, The limited in vitro lifetime of human diploid cell strains.Exp. Cell Res.37, 614–636.CrossRefGoogle Scholar
  37. 37.
    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 and Stem Cells3, 221–231.CrossRefGoogle Scholar
  38. 38.
    Lanza, R.P., Cibelli, J.B., Blackwell, C., Cristofalo, V.J., Francis, M.K., Baerlocher, G.M., Mak, J., Schertzer, M., Chavez, E.A., Sawyer, N., Lansdorp, P.M., and West, M.D., 2000, Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.Science288, 665–669.CrossRefGoogle Scholar
  39. 39.
    Cristofalo, V.J., and Pignolo, R.J., 1996, Molecular markers of senescence in fibroblast-like cultures.Exp. Gerontol.31, 111–123.CrossRefGoogle Scholar
  40. 40.
    Frippiat, C., Chen, Q.M., Zdanov, S., Magalhaes, J-P., Remade, J., and Toussain, O., 2001, Subcytotoxic H2O2stress triggers a release of transforming growth factor-El, which induces biomarkers of cellular senescence of human diploid fibroblasts.J. Biol. Chem.276, 2531–2537.CrossRefGoogle Scholar
  41. 41.
    Sedivy JM., 1998, Can ends justify the means?: telomeres and the mechanisms of replicative senescence and immortalization in mammalian cells.Proc. Natl. Acad. Sci. USA95, 9078–9081.CrossRefGoogle Scholar
  42. 42.
    Rubin H., 2002, The disparity between human cell senescence in vitro and lifelong replication in vivo.Nature Biotechnol.20, 675–681.CrossRefGoogle Scholar
  43. 43.
    Kubota, C., Yamakuchi, H., Todoroki, J., Mizoshita, K., Tabara, N., Barber, M., and Yang, X., 2000, Six cloned calves produced from adult fibroblast cells after long-term culture.Proc. Natl. Acad. Sci. USA97, 990–995CrossRefGoogle Scholar
  44. 44.
    Thomson, A.J., Marques, M.M., and McWhir, J., 2002, Gene targeting in livestock.Reproduction(in press).Google Scholar
  45. 45.
    Bodnar, A.G., Oullette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., Lichtsteiner, S., and Wright, W.E., 1998, Extension of life-span by introduction of telomerase into normal human cells.Science279, 349–352.CrossRefGoogle Scholar
  46. 46.
    Rubio, M.A., Kim, S-H., and Campisi, J., 2002, Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells.J. Biol. Chem.277, 28609–28617.CrossRefGoogle Scholar
  47. 47.
    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, 38531–38539.CrossRefGoogle Scholar
  48. 48.
    Cibelli, J.B., Stice, S.L., Golueke, P.J., Kane, J.F., Jerry, J., Blackwell, C., Ponce de León, A., and Robl, J.M., 1998, Cloned transgenic calves produced from nonquiescent fetal fibroblasts.Science280, 1256–1258.CrossRefGoogle Scholar
  49. 49.
    Sedivy, J.M., and Dutriaux, A., 1999, Gene targeting and somatic cell genetics: a rebirth or a coming of age?.TIG15, 88–90.CrossRefGoogle Scholar
  50. 50.
    Marques, M.M., Thomson, A.J., McCreath, K.J., and McWhir, J., (manuscript in preparation).Google Scholar
  51. 51.
    Thomson, A.J., Marques, M.M., and McWhir, J., (manuscript in preparation).Google Scholar
  52. 52.
    Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A., and Kucherlapatti, R.S., 1985, Insertion of DNA sequences into the human chromosomal E-globin locus by homologous recombination.Nature317, 230–234.CrossRefGoogle Scholar
  53. 53.
    Felsenfeld, G., Boyes, J., Chung, J., Clark, D., and Studitsky, V., 1996, Chromatin structure and gene expression.Proc. Natl. Acad. Sci. USA93, 9384–9388.CrossRefGoogle Scholar
  54. 54.
    Gregory, P.D., and Horz, W., 1998, Chromatin and transcription-how transcription factors battle with a repressive chromatin environment.Eur. J. Biochem.251, 9–18.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2003

Authors and Affiliations

  • Margarita M. Marques
    • 1
  • Alison J. Thomson
  • Jim McWhir
  1. 1.Department of Gene Expression and Development, Roslin InstituteRoslin, MidlothianScotland, UK

Personalised recommendations