Somatic Cell Nuclear Transfer and the Creation of Transgenic Large Animal Models

  • Naomi Dicks
  • Luis B. Agellon
  • Vilceu Bordignon


Transgenic animals have been instrumental in biomedical and genetic research, particularly through the use of genetically modified mice. To facilitate translational research to humans, the development of larger species of transgenic animals is necessary. Furthermore, these transgenic large animal models have numerous other applications in biomedicine and agriculture. Various techniques to produce genetically altered animals have been developed throughout the years including pronuclear microinjection, sperm-mediated gene transfer (SMGT), electroporation, oocyte/embryo transduction and somatic cell nuclear transfer (SCNT). A major advantage of the SCNT technique is the ability to genetically modify donor cells and select for these transgenic cells prior to the cloning procedure, resulting in 100 % transgenic efficiency. This has particularly facilitated the production of transgenic large animals, for which embryonic stem cells that can be cultured and reliably modified in the laboratory are not fully established. In addition, new gene-editing technologies, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have further assisted the creation of transgenic animals, notably gene-disrupted animals. Demand for these large transgenic animal models is increasing as a result of their numerous possible applications including the development of higher quality production animals, creation of stem cells for tissue repair (therapeutic cloning), production of protein-based pharmaceuticals (animal pharming), creation of organ donors for xenotransplantation and the creation of large animal models for biomedical research.


Animal cloning Gene-editing RNA Interference Transcription activator-like effector nucleases Zinc finger nucleases 


  1. Bordignon V (2011) Animal systems animal cloning: state of the art and applications. In: Murray M-Y (ed) Comprehensive biotechnology, vol 4. 4441–4456Google Scholar
  2. Bordignon V, El-Beirouthi N, Gasperin BG, Albornoz MS, Martinez-Diaz MA, Schneider C, Laurin D, Zadworny D, Agellon LB (2013) Production of cloned pigs with targeted attenuation of gene expression. PLoS ONE 8:e64613CrossRefPubMedCentralPubMedGoogle Scholar
  3. Briggs R, King TJ (1952) Transplantation of living nuclei from blastula cells into enucleated frogs’ eggs. Proc Natl Acad Sci U S A 38:455–463CrossRefPubMedCentralPubMedGoogle Scholar
  4. Cabot RA, Kuhholzer B, Chan AW, Lai L, Park KW, Chong KY, Schatten G, Murphy CN, Abeydeera LR, Day BN, Prather RS (2001) Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Anim Biotechnol 12:205–214CrossRefPubMedGoogle Scholar
  5. Campbell KH, Alberio R, Choi I, Fisher P, Kelly RD, Lee JH, Maalouf W (2005) Cloning: eight years after Dolly. Reprod Domes Anim 40:256–268CrossRefGoogle Scholar
  6. Campbell KH, Fisher P, Chen WC, Choi I, Kelly RD, Lee JH, Xhu J (2007). Somatic cell nuclear transfer: past, present and future perspectives. Theriogenology 68(Suppl 1):214–31CrossRefGoogle Scholar
  7. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, Voytas DF, Long CR, Whitelaw CB, Fahrenkrug SC (2012) Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci U S A 109:17382–17387CrossRefPubMedCentralPubMedGoogle Scholar
  8. Chang N, Sun C, Gao L, Zhu D, Xu X, Zhu X, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos. Cell Res 23:465–472CrossRefPubMedCentralPubMedGoogle Scholar
  9. Cho B, Koo OJ, Hwang JI, Kim H, Lee EM, Hurh S, Park SJ, Ro H, Yang J, Surh CD, D’Apice AJ, Lee BC, Ahn C (2011) Generation of soluble human tumor necrosis factor-alpha receptor 1-Fc transgenic pig. Transplantation 92:139–147CrossRefPubMedGoogle Scholar
  10. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823CrossRefPubMedCentralPubMedGoogle Scholar
  11. Echelard Y, Williams JL, Destrempes MM, Koster JA, Overton SA, Pollock DP, Rapiejko KT, Behboodi E, Masiello NC, Gavin WG, Pommer J, Van Patten SM, Faber DC, Cibelli JB, Meade HM (2009) Production of recombinant albumin by a herd of cloned transgenic cattle. Transgenic Res 18:361–376CrossRefPubMedGoogle Scholar
  12. Edwards JL, Schrick FN, McCracken MD, van Amstel SR, Hopkins FM, Welborn MG, Davies CJ (2003) Cloning adult farm animals: a review of the possibilities and problems associated with somatic cell nuclear transfer. Am J Reprod Immunol 50:113–123CrossRefPubMedGoogle Scholar
  13. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in. Caenorhabditis elegans Nature 391:806–811CrossRefGoogle Scholar
  14. Gordon JW, Ruddle FH (1981) Integration and stable germ line transmission of genes injected into mouse pronuclei. Science 214:1244–1246CrossRefPubMedGoogle Scholar
  15. Hannon GJ, Rossi JJ (2004) Unlocking the potential of the human genome with RNA interference. Nature 431:371–378CrossRefPubMedGoogle Scholar
  16. Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann H (2011) Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci U S A 108:12013–12017CrossRefPubMedCentralPubMedGoogle Scholar
  17. Ibrahim Z, Busch J, Awwad M, Wagner R, Wells K, Cooper DK (2006) Selected physiologic compatibilities and incompatibilities between human and porcine organ systems. Xenotransplantation 13:488–499CrossRefPubMedGoogle Scholar
  18. Illmensee K, Hoppe PC (1981) Nuclear transplantation in Mus musculus: developmental potential of nuclei from preimplantation embryos. Cell 23:9–18CrossRefPubMedGoogle Scholar
  19. Jabed A, Wagner S, McCracken J, Wells DN, Laible G (2012) Targeted microRNA expression in dairy cattle directs production of beta-lactoglobulin-free, high-casein milk. Proc Natl Acad Sci U S A 109:16811–16816CrossRefPubMedCentralPubMedGoogle Scholar
  20. Jang G, Kim MK, Lee BC (2010) Current status and applications of somatic cell nuclear transfer in dogs. Theriogenology 74:1311–1320CrossRefPubMedGoogle Scholar
  21. Kashiwakura Y, Mimuro J, Onishi A, Iwamoto M, Madoiwa S, Fuchimoto D, Suzuki S, Suzuki M, Sembon S, Ishiwata A, Yasumoto A, Sakata A, Ohmori T, Hashimoto M, Yazaki S, Sakata Y (2012) Porcine model of hemophilia A. PLoS ONE 7:e49450CrossRefPubMedCentralPubMedGoogle Scholar
  22. Lai L, Sun Q, Wu G, Murphy CN, Kuhholzer B, Park KW, Bonk AJ, Day BN, Prather RS (2001) Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes. Zygote 9:339–346CrossRefPubMedGoogle Scholar
  23. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science 295:1089–1092CrossRefPubMedGoogle Scholar
  24. Lai L, Kang JX, Li R, Wang J, Witt WT, Yong HY, Hao Y, Wax DM, Murphy CN, Rieke A, Samuel M, Linville ML, Korte SW, Evans RW, Starzl TE, Prather RS, Dai Y (2006) Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nat Biotechnol 24:435–436CrossRefPubMedCentralPubMedGoogle Scholar
  25. Lavitrano M, Forni M, Bacci ML, Di Stefano C, Varzi V, Wang H, Seren E (2003) Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Mol Reprod Dev 64:284–291CrossRefPubMedGoogle Scholar
  26. Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B (2011) TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res 39:359–372CrossRefPubMedCentralPubMedGoogle Scholar
  27. Luo Y, Lin L, Bolund L, Jensen TG, Sorensen CB (2012) Genetically modified pigs for biomedical research. J Inherit Metab Dis 35:695–713CrossRefPubMedGoogle Scholar
  28. Maksimenko OG, Deykin AV, Khodarovich YM, Georgiev PG (2013) Use of transgenic animals in biotechnology: prospects and problems. Acta Naturae 5:33–46PubMedCentralPubMedGoogle Scholar
  29. Mallo M (2006) Controlled gene activation and inactivation in the mouse. Front Biosci 11:313–327CrossRefPubMedGoogle Scholar
  30. Miller J, McLachlan AD, Klug A (1985) Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes. EMBO J 4:1609–1614PubMedCentralPubMedGoogle Scholar
  31. Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res 39:9283–9293CrossRefPubMedCentralPubMedGoogle Scholar
  32. Niemann H, Lucas-Hahn A (2012) Somatic cell nuclear transfer cloning: practical applications and current legislation. Reprod Domes Anim 47(Suppl 5):2–10CrossRefGoogle Scholar
  33. Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang AP, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J (2014). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156:836–43CrossRefPubMedGoogle Scholar
  34. Oropeza M, Petersen B, Carnwath JW, Lucas-Hahn A, Lemme E, Hassel P, Herrmann D, Barg-Kues B, Holler S, Queisser AL, Schwinzer R, Hinkel R, Kupatt C, Niemann H (2009). Transgenic expression of the human A20 gene in cloned pigs provides protection against apoptotic and inflammatory stimuli. Xenotransplantation 16:522–34CrossRefPubMedGoogle Scholar
  35. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958CrossRefPubMedCentralPubMedGoogle Scholar
  36. Pan D, Zhang L, Zhou Y, Feng C, Long C, Liu X, Wan R, Zhang J, Lin A, Dong E, Wang S, Xu H, Chen H (2010) Efficient production of omega-3 fatty acid desaturase (sFat-1)-transgenic pigs by somatic cell nuclear transfer. Sci China Life Sci 53:517–523CrossRefPubMedGoogle Scholar
  37. Pereyra-Bonnet F, Fernandez-Martin R, Olivera R, Jarazo J, Vichera G, Gibbons A, Salamone D (2008) A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species. Reprod Fertil Dev 20:741–749CrossRefPubMedGoogle Scholar
  38. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL (2003) Production of alpha 1,3-galactosyltransferase-deficient pigs. Science 299:411–414CrossRefPubMedCentralPubMedGoogle Scholar
  39. Phelps CJ, Ball SF, Vaught TD, Vance AM, Mendicino M, Monahan JA, Walters AH, Wells KD, Dandro AS, Ramsoondar JJ, Cooper DK, Ayares DL (2009) Production and characterization of transgenic pigs expressing porcine CTLA4-Ig. Xenotransplantation 16:477–485CrossRefPubMedGoogle Scholar
  40. Prather RS, Lorson M, Ross JW, Whyte JJ, Walters E (2013) Genetically engineered pig models for human diseases. Annu Rev Anim Biosci 1:203–219CrossRefPubMedGoogle Scholar
  41. Ramsoondar J, Vaught T, Ball S, Mendicino M, Monahan J, Jobst P, Vance A, Duncan J, Wells K, Ayares D (2009) Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation 16:164–180CrossRefPubMedGoogle Scholar
  42. Redwan el RM (2009) Animal-derived pharmaceutical proteins. J Immunoass Immunochem 30:262–290CrossRefGoogle Scholar
  43. Renner S, Fehlings C, Herbach N, Hofmann A, von Waldthausen DC, Kessler B, Ulrichs K, Chodnevskaja I, Moskalenko V, Amselgruber W, Goke B, Pfeifer A, Wanke R, Wolf E (2010) Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function. Diabetes 59:1228–1238CrossRefPubMedCentralPubMedGoogle Scholar
  44. Renner S, Braun-Reichhart C, Blutke A, Herbach N, Emrich D, Streckel E, Wunsch A, Kessler B, Kurome M, Bahr A, Klymiuk N, Krebs S, Puk O, Nagashima H, Graw J, Blum H, Wanke R, Wolf E (2013) Permanent neonatal diabetes in INSC94Y transgenic pigs. Diabetes 62:1505–1511CrossRefPubMedCentralPubMedGoogle Scholar
  45. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB Jr, Zabner J, Prather RS, Welsh MJ (2008) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321:1837–1841CrossRefPubMedCentralPubMedGoogle Scholar
  46. Salamone D, Baranao L, Santos C, Bussmann L, Artuso J, Werning C, Prync A, Carbonetto C, Dabsys S, Munar C, Salaberry R, Berra G, Berra I, Fernandez N, Papouchado M, Foti M, Judewicz N, Mujica I, Munoz L, Alvarez SF, Gonzalez E, Zimmermann J, Criscuolo M, Melo C (2006) High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow. J Biotechnol 124:469–472CrossRefPubMedGoogle Scholar
  47. Sato M, Miyoshi K, Nagao Y, Nishi Y, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S (2014) The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3-galactosyltransferase gene in porcine embryonic fibroblasts. Xenotransplantation 21:291–300CrossRefPubMedGoogle Scholar
  48. Takahagi Y, Fujimura T, Miyagawa S, Nagashima H, Shigehisa T, Shirakura R, Murakami H (2005) Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III. Mol Reprod Dev 71:331–338CrossRefPubMedGoogle Scholar
  49. Umeyama K, Watanabe M, Saito H, Kurome M, Tohi S, Matsunari H, Miki K, Nagashima H (2009) Dominant-negative mutant hepatocyte nuclear factor 1alpha induces diabetes in transgenic-cloned pigs. Transgenic Res 18:697–706CrossRefPubMedGoogle Scholar
  50. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646CrossRefPubMedGoogle Scholar
  51. Vajta G (2007) Handmade cloning: the future way of nuclear transfer? Trends Biotechnol 25:250–253CrossRefPubMedGoogle Scholar
  52. Wall RJ (2001) Pronuclear microinjection. Cloning Stem Cells 3:209–220CrossRefPubMedGoogle Scholar
  53. Wall RJ, Powell AM, Paape MJ, Kerr DE, Bannerman DD, Pursel VG, Wells KD, Talbot N, Hawk HW (2005) Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nat Biotechnol 23:445–451CrossRefPubMedGoogle Scholar
  54. Wang B, Zhou J (2003) Specific genetic modifications of domestic animals by gene targeting and animal cloning. Reprod Biol Endocrinol 103:1–8Google Scholar
  55. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910–918CrossRefPubMedCentralPubMedGoogle Scholar
  56. Watanabe M, Kurome M, Matsunari H, Nakano K, Umeyema K, Shiota A, Nakauchi H, Nagashima H (2012) The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer. Transgenic Res 21:605–618CrossRefPubMedGoogle Scholar
  57. Watt FM, Driskell RR (2010) The therapeutic potential of stem cells. Philos Trans R Soc Lond B Biol Sci 365:155–163CrossRefPubMedCentralPubMedGoogle Scholar
  58. Wei J, Ouyang H, Wang Y, Pang D, Cong NX, Wang T, Leng B, Li D, Li X, Wu R, Ding Y, Gao F, Deng Y, Liu B, Li Z, Lai L, Feng H, Liu G, Deng X (2012) Characterization of a hypertriglyceridemic transgenic miniature pig model expressing human apolipoprotein CIII. FEBS J 279:91–99CrossRefPubMedGoogle Scholar
  59. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O’Gorman C, Walters EM, Murphy CN, Driver JP, Mileham A, McLaren D, Wells KD, Prather RS (2014). Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91(3):78. pii: biolreprod.114.121723CrossRefPubMedGoogle Scholar
  60. Whyte JJ, Prather RS (2012) Cell biology symposium: Zinc finger nucleases to create custom-designed modifications in the swine (Sus scrofa) genome. J Anim Sci 90:1111–1117CrossRefPubMedGoogle Scholar
  61. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385:810–813CrossRefPubMedGoogle Scholar
  62. Wu X, Ouyang H, Duan B, Pang D, Zhang L, Yuan T, Xue L, Ni D, Cheng L, Dong S, Wei Z, Li L, Yu M, Sun QY, Chen DY, Lai L, Dai Y, Li GP (2012) Production of cloned transgenic cow expressing omega-3 fatty acids. Transgenic Res 21:537–543CrossRefPubMedGoogle Scholar
  63. Yamanaka S, Takahashi K (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2015

Authors and Affiliations

  1. 1.Department of Animal ScienceMcGill UniversitySte-Anne-de-BellevueCanada
  2. 2.School of Dietetics and Human NutritionMcGill UniversitySte-Anne-de-BellevueCanada
  3. 3.Department of Animal ScienceMcGill UniversitySte-Anne-de-BellevueCanada

Personalised recommendations