Transgenic Research

, Volume 20, Issue 5, pp 1125–1137 | Cite as

Strategies for selection marker-free swine transgenesis using the Sleeping Beauty transposon system

  • Daniel F. Carlson
  • John R. Garbe
  • Wenfang Tan
  • Mike J. Martin
  • John R. Dobrinsky
  • Perry B. Hackett
  • Karl J. Clark
  • Scott C. Fahrenkrug
Original Paper

Abstract

Swine transgenesis by pronuclear injection or cloning has traditionally relied on illegitimate recombination of DNA into the pig genome. This often results in animals containing concatemeric arrays of transgenes that complicate characterization and can impair long-term transgene stability and expression. This is inconsistent with regulatory guidance for transgenic livestock, which also discourages the use of selection markers, particularly antibiotic resistance genes. We demonstrate that the Sleeping Beauty (SB) transposon system effectively delivers monomeric, multi-copy transgenes to the pig embryo genome by pronuclear injection without markers, as well as to donor cells for founder generation by cloning. Here we show that our method of transposon-mediated transgenesis yielded 38 cloned founder pigs that altogether harbored 100 integrants for five distinct transposons encoding either human APOBEC3G or YFP-Cre. Two strategies were employed to facilitate elimination of antibiotic genes from transgenic pigs, one based on Cre-recombinase and the other by segregation of independently transposed transgenes upon breeding.

Keywords

Swine transgenesis Sleeping Beauty Transposon Cre recombinase Cloning Pronuclear injection 

Notes

Acknowledgments

The authors would like to thank Sandra Horn from the Mouse Genetics lab at the University of Minnesota for help with pig pronuclear injection. We thank Steve Terlouw, Bradley Didion, Richard Koppang, Ann Marie Paprocki, Charity Syverson, Greg Mell, Gordy Gunderson and Eric James for pig cloning, founder management and sample collection and Ludwig and Becky Simmet for their generous support. The authors would also like to thank Zsuzsanna Izsvák, Zoltán Ivics and Lajos Mátés for providing SB100X. This project was supported by National Research Initiative Competitive Grant no. 2008-35205-18852 from the USDA National Institute of Food and Agriculture.

Supplementary material

11248_2010_9481_MOESM1_ESM.doc (570 kb)
Supplementary material 1 (DOC 570 kb)

References

  1. Abuin A, Bradley A (1996) Recycling selectable markers in mouse embryonic stem cells. Mol Cell Biol 16(4):1851–1856PubMedGoogle Scholar
  2. Carlson DF, Geurts AM, Garbe JR, Park CW, Rangel-Filho A, O’Grady SM, Jacob HJ, Steer CJ, Largaespada DA, Fahrenkrug SC (2010) Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Res. March 30, Epub ahead of printGoogle Scholar
  3. Carter DB, Lai L, Park KW, Samuel M, Lattimer JC, Jordan KR, Estes DM, Besch-Williford C, Prather RS (2002) Phenotyping of transgenic cloned piglets. Cloning Stem Cells 4(2):131–145PubMedCrossRefGoogle Scholar
  4. Chen YT, Bradley A (2000) A new positive/negative selectable marker, puDeltatk, for use in embryonic stem cells. Genesis 28(1):31–35PubMedCrossRefGoogle Scholar
  5. Chen CM, Choo KB, Cheng WT (1995) Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences. Transgenic Res 4(1):52–59PubMedCrossRefGoogle Scholar
  6. Clark KJ, Carlson DF, Fahrenkrug SC (2007a) Pigs taking wing with transposons and recombinases. Genome biol 8(Suppl 1):S13Google Scholar
  7. Clark KJ, Carlson DF, Foster LK, Kong BW, Foster DN, Fahrenkrug SC (2007b) Enzymatic engineering of the porcine genome with transposons and recombinases. BMC biotech 7:42CrossRefGoogle Scholar
  8. Clark KJ, Carlson DF, Leaver MJ, Foster LK, Fahrenkrug SC (2009) Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells. Nucleic Acids Res 37(4):1239–1247PubMedCrossRefGoogle Scholar
  9. Codex Alimentarius (2003) Guidline for the conduct of food safety assessment of foods derived from recombinant-DNA plants (CAC/GL 45-2003)Google Scholar
  10. Codex Alimentarius (2008) Report of the seventh session of the codex Ad Hoc Intergovernmental task force on foods derived from biotechnology (ALINORM 08/31/34)Google Scholar
  11. Collas P, Robl JM, Sullivan E, Kasinathan P (2007) Methods for cloning non-human mammals using reprogrammed donor chromatin or donor cells. US Patent 7,253,334Google Scholar
  12. Covarrubias L, Nishida Y, Mintz B (1986) Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain. Proc Natl Acad Sci USA 83(16):6020–6024PubMedCrossRefGoogle Scholar
  13. Dalle B, Rubin JE, Alkan O, Sukonnik T, Pasceri P, Yao S, Pawliuk R, Leboulch P, Ellis J (2005) eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides. Mol Ther 11(4):591–599PubMedCrossRefGoogle Scholar
  14. de Alboran IM, O’Hagan RC, Gartner F, Malynn B, Davidson L, Rickert R, Rajewsky K, DePinho RA, Alt FW (2001) Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity 14(1):45–55PubMedCrossRefGoogle Scholar
  15. Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122(3):473–483PubMedCrossRefGoogle Scholar
  16. Dorer DR, Henikoff S (1997) Transgene repeat arrays interact with distant heterochromatin and cause silencing in cis and trans. Genetics 147(3):1181–1190PubMedGoogle Scholar
  17. Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, Markley KM, Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn S, Largaespada DA (2002) Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci USA 99(7):4495–4499PubMedCrossRefGoogle Scholar
  18. FDA: Guidance for Industry (2009) Regulation of Genetically Engineered AnimalsContaining Heritable Recombinant DNA ConstructsGoogle Scholar
  19. Garrick D, Fiering S, Martin DI, Whitelaw E (1998) Repeat-induced gene silencing in mammals. Nat Genet 18(1):56–59PubMedCrossRefGoogle Scholar
  20. Geurts AM, Collier LS, Geurts JL, Oseth LL, Bell ML, Mu D, Lucito R, Godbout SA, Green LE, Lowe SW, Hirsch BA, Leinwand LA, Largaespada DA (2006) Gene mutations and genomic rearrangements in the mouse as a result of transposon mobilization from chromosomal concatemers. PLoS Genet 2(9):e156PubMedCrossRefGoogle Scholar
  21. Gordon JW, Ruddle FH (1985) DNA-mediated genetic transformation of mouse embryos and bone marrow–a review. Gene 33(2):121–136PubMedCrossRefGoogle Scholar
  22. Grabundzija I, Irgang M, Mates L, Belay E, Matrai J, Gogol-Doring A, Kawakami K, Chen W, Ruiz P, Chuah MK, VandenDriessche T, Izsvak Z, Ivics Z (2010) Comparative analysis of transposable element vector systems in human cells. Mol Ther 18(6):1200–1209PubMedCrossRefGoogle Scholar
  23. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL (1985) Production of transgenic rabbits, sheep and pigs by microinjection. Nature 315(6021):680–683PubMedCrossRefGoogle Scholar
  24. Hofmann A, Kessler B, Ewerling S, Weppert M, Vogg B, Ludwig H, Stojkovic M, Boelhauve M, Brem G, Wolf E, Pfeifer A (2003) Efficient transgenesis in farm animals by lentiviral vectors. EMBO Rep 4(11):1054–1060PubMedCrossRefGoogle Scholar
  25. Hofmann A, Zakhartchenko V, Weppert M, Sebald H, Wenigerkind H, Brem G, Wolf E, Pfeifer A (2004) Generation of transgenic cattle by lentiviral gene transfer into oocytes. Biol Reprod 71(2):405–409PubMedCrossRefGoogle Scholar
  26. Jakobsen JE, Li J, Kragh PM, Moldt B, Lin L, Liu Y, Schmidt M, Winther KD, Schyth BD, Holm IE, Vajta G, Bolund L, Callesen H, Jorgensen AL, Nielsen AL, Mikkelsen JG (2010a) Pig transgenesis by Sleeping Beauty DNA transposition. Transgenic Res. Aug 29, Epub ahead of printGoogle Scholar
  27. Jakobsen JE, Li J, Moldt B, Kragh PM, Callesen H, Hertz JM, Bolund L, Jorgensen AL, Mikkelsen JG, Nielsen AL (2010b) Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts. Mol Biol Rep. March 25, Epub ahead of printGoogle Scholar
  28. Jonsson SR, LaRue RS, Stenglein MD, Fahrenkrug SC, Andresdottir V, Harris RS (2007) The restriction of zoonotic PERV transmission by human APOBEC3G. PloS one 2(9):e893PubMedCrossRefGoogle Scholar
  29. Krumlauf R, Hammer RE, Tilghman SM, Brinster RL (1985) Developmental regulation of alpha-fetoprotein genes in transgenic mice. Mol Cell Biol 5(7):1639–1648PubMedGoogle Scholar
  30. Lewandoski M (2001) Conditional control of gene expression in the mouse. Nat Rev Genet 2(10):743–755PubMedCrossRefGoogle Scholar
  31. Long CR, Dobrinsky JR, Johnson LA (1999) In vitro production of pig embryos: comparison of culture media and boars. Theriogenology 51:1375–1390PubMedCrossRefGoogle Scholar
  32. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R, Berns A, Jonkers J (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA 98(16):9209–9214PubMedCrossRefGoogle Scholar
  33. Mark WH, Signorelli K, Blum M, Kwee L, Lacy E (1992) Genomic structure of the locus associated with an insertional mutation in line 4 transgenic mice. Genomics 13(1):159–166PubMedCrossRefGoogle Scholar
  34. Mátés L, Chuah MKL, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Judis C, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Mathieu C, Pryputniewicz D, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z (2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41(6):753–761PubMedCrossRefGoogle Scholar
  35. Nakanishi T, Kuroiwa A, Yamada S, Isotani A, Yamashita A, Tairaka A, Hayashi T, Takagi T, Ikawa M, Matsuda Y, Okabe M (2002) FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 80(6):564–574PubMedCrossRefGoogle Scholar
  36. Ornitz DM, Palmiter RD, Hammer RE, Brinster RL, Swift GH, MacDonald RJ (1985) Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice. Nature 313(6003):600–602PubMedCrossRefGoogle Scholar
  37. Overbeek PA, Lai SP, Van Quill KR, Westphal H (1986) Tissue-specific expression in transgenic mice of a fused gene containing RSV terminal sequences. Science 231(4745):1574–1577PubMedCrossRefGoogle Scholar
  38. Palmiter RD, Norstedt G, Gelinas RE, Hammer RE, Brinster RL (1983) Metallothionein-human GH fusion genes stimulate growth of mice. Science 222(4625):809–814PubMedCrossRefGoogle Scholar
  39. Pravtcheva DD, Wise TL (1995) A postimplantation lethal mutation induced by transgene insertion on mouse chromosome 8. Genomics 30(3):529–544PubMedCrossRefGoogle Scholar
  40. Pravtcheva DD, Wise TL (2003) Transgene instability in mice injected with an in vitro methylated Igf2 gene. Mutat Res Fundam Mol Mech Mutagen 529(1–2):35–50CrossRefGoogle Scholar
  41. Ritchie WA, King T, Neil C, Carlisle AJ, Lillico S, McLachlan G, Whitelaw CB (2009) Transgenic sheep designed for transplantation studies. Mol Reprod Dev 76(1):61–64PubMedCrossRefGoogle Scholar
  42. Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, Petroff E, Vermeer DW, Kabel AC, Yan Z, Spate L, Wax D, Murphy CN, Rieke A, Whitworth K, Linville ML, Korte SW, Engelhardt JF, Welsh MJ, Prather RS (2008) Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. J Clin Invest 118(4):1571–1577PubMedCrossRefGoogle Scholar
  43. Rohan RM, King D, Frels WI (1990) Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes. Nucleic Acids Res 18(20):6089–6095PubMedCrossRefGoogle Scholar
  44. Schmidt EE, Taylor DS, Prigge JR, Barnett S, Capecchi MR (2000) Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA 97(25):13702–13707PubMedCrossRefGoogle Scholar
  45. Scrable H, Stambrook PJ (1999) A genetic program for deletion of foreign DNA from the mammalian genome. Mutat Res 429(2):225–237PubMedGoogle Scholar
  46. Townes TM, Chen HY, Lingrel JB, Palmiter RD, Brinster RL (1985) Expression of human beta-globin genes in transgenic mice: effects of a flanking metallothionein-human growth hormone fusion gene. Mol Cell Biol 5(8):1977–1983PubMedGoogle Scholar
  47. Urschitz J, Kawasumi M, Owens J, Morozumi K, Yamashiro H, Stoytchev I, Marh J, Dee JA, Kawamoto K, Coates CJ, Kaminski JM, Pelczar P, Yanagimachi R, Moisyadi S (2010) Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl Acad Sci USA 107(18):8117–8122PubMedCrossRefGoogle Scholar
  48. Wilber A, Frandsen JL, Wangensteen KJ, Ekker SC, Wang X, McIvor RS (2005) Dynamic gene expression after systemic delivery of plasmid DNA as determined by in vivo bioluminescence imaging. Hum Gene Ther 16(11):1325–1332PubMedCrossRefGoogle Scholar
  49. Wilber A, Frandsen JL, Geurts JL, Largaespada DA, Hackett PB, McIvor RS (2006) RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther 13(3):625–630PubMedCrossRefGoogle Scholar
  50. Wilkie TM, Palmiter RD (1987) Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant. Mol Cell Biol 7(5):1646–1655PubMedGoogle Scholar
  51. Yusa K, Takeda J, Horie K (2004) Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation. Mol Cell Biol 24(9):4004–4018PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Daniel F. Carlson
    • 1
    • 2
  • John R. Garbe
    • 2
  • Wenfang Tan
    • 1
    • 2
  • Mike J. Martin
    • 3
  • John R. Dobrinsky
    • 4
  • Perry B. Hackett
    • 1
    • 5
  • Karl J. Clark
    • 7
  • Scott C. Fahrenkrug
    • 1
    • 2
    • 6
  1. 1.The Center for Genome EngineeringUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of Animal ScienceUniversity of MinnesotaSaint PaulUSA
  3. 3.Spring Point ProjectMinneapolisUSA
  4. 4.Minitube Biotechnology CenterMt. HorebUSA
  5. 5.Department of Genetics, Cell Biology and DevelopmentMinneapolisUSA
  6. 6.RecombineticsMinneapolisUSA
  7. 7.Department of Biochemistry and Molecular BiologyMayo ClinicRochesterUSA

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