Transgenic Research

, Volume 27, Issue 2, pp 167–178 | Cite as

Zygote injection of RNA encoding Cre recombinase results in efficient removal of LoxP flanked neomycin cassettes in pigs

  • Kristin M. Whitworth
  • Raissa Cecil
  • Joshua A. Benne
  • Bethany K. Redel
  • Lee D. Spate
  • Melissa S. Samuel
  • Randall S. Prather
  • Kevin D. Wells
Original Paper


Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre–loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/μL when compared to 2 and 100 ng/μL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/μL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.


Cre–loxP Pig zygote Somatic cell nuclear transfer CD163 



The study was funded by University of Missouri, Food for the 21st Century.


  1. Bauer BK, Spate LD, Murphy CN, Prather RS (2010) 1 Arginine supplementation in vitro increases porcine embryo development and affects mrna transcript expression. Reprod Fertil Dev 23(1):107CrossRefGoogle Scholar
  2. Beaton BP, Wells KD (2014) Compound transgenics: recombinase-mediated gene stacking. Elsevier, Amsterdam, pp 565–578Google Scholar
  3. Bi Y, Hua Z, Liu X, Hua W, Ren H, Xiao H, Zhang L, Li L, Wang Z, Laible G, Wang Y, Dong F, Zheng X (2016) Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP. Sci Rep 6:31729CrossRefPubMedPubMedCentralGoogle Scholar
  4. Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6(1):7–28CrossRefPubMedGoogle Scholar
  5. Bressan FF, Miranda MS, Bajgelman MC, Perecin F, Mesquita LG, Fantinato-Neto P, Merighe GF, Strauss BE, Meirelles FV (2013) Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer. In Vitro Cell Dev Biol Anim 49(4):250–259CrossRefPubMedGoogle Scholar
  6. Burkard C, Lillico SG, Reid E, Jackson B, Mileham AJ, Ait-Ali T, Whitelaw CB, Archibald AL (2017) Precision engineering for PRRSV resistance in pigs: macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLoS Pathog 13(2):e1006206CrossRefPubMedPubMedCentralGoogle Scholar
  7. 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):131–145CrossRefPubMedGoogle Scholar
  8. Chen L, Li L, Pang D, Li Z, Wang T, Zhang M, Song N, Yan S, Lai LX, Ouyang H (2010) Construction of transgenic swine with induced expression of Cre recombinase. Anim Int J Anim Biosci 4(5):767–771CrossRefGoogle Scholar
  9. Cho J, Bhuiyan MM, Shin S, Park E, Jang G, Kang S, Lee B, Hwang W (2004) Development potential of transgenic somatic cell nuclear transfer embryos according to various factors of donor cell. J Vet Med Sci 66(12):1567–1573CrossRefPubMedGoogle Scholar
  10. Ekser B, Li P, Cooper DKC (2017) Xenotransplantation: past, present, and future. Curr Opin Organ Transpl 22(6):513–521Google Scholar
  11. Gupta N, Taneja R, Pandey A, Mukesh M, Singh H, Gupta SC (2007) Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat’s skin fibroblast cell line. Cell Biol Int 31(10):1257–1264CrossRefPubMedGoogle Scholar
  12. Hagen DR, Prather RS, Sims MM, First NL (1991) Development of one-cell porcine embryos to the blastocyst stage in simple media. J Anim Sci 69(3):1147–1150CrossRefPubMedGoogle Scholar
  13. Han XJ, Liang H, Yun T, Zhao YH, Zhang ML, Zhao LH, Li RF, Li XL (2015) Decreased expression of humanized Fat-1 in porcine fetal fibroblasts following deletion of PGK-neomycin resistance. Genet Mol Res GMR 14(3):11594–11604CrossRefPubMedGoogle Scholar
  14. Hoess RH, Abremski K (1984) Interaction of the bacteriophage P1 recombinase Cre with the recombining site loxP. Proc Natl Acad Sci U S A 81(4):1026–1029CrossRefPubMedPubMedCentralGoogle Scholar
  15. Hoess RH, Ziese M, Sternberg N (1982) P1 site-specific recombination: nucleotide sequence of the recombining sites. Proc Natl Acad Sci U S A 79(11):3398–3402CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jensen TW, Mazur MJ, Pettigew JE, Perez-Mendoza VG, Zachary J, Schook LB (2010) A cloned pig model for examining atherosclerosis induced by high fat, high cholesterol diets. Anim Biotechnol 21(3):179–187CrossRefPubMedGoogle Scholar
  17. Kolber-Simonds D, Lai L, Watt SR, Denaro M, Arn S, Augenstein ML, Betthauser J, Carter DB, Greenstein JL, Hao Y, Im GS, Liu Z, Mell GD, Murphy CN, Park KW, Rieke A, Ryan DJ, Sachs DH, Forsberg EJ, Prather RS, Hawley RJ (2004) Production of alpha-1,3-galactosyltransferase null pigs by means of nuclear transfer with fibroblasts bearing loss of heterozygosity mutations. Proc Natl Acad Sci U S A 101(19):7335–7340CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kurome M, Hisatomi H, Matsumoto S, Tomii R, Ueno S, Hiruma K, Saito H, Nakamura K, Okumura K, Matsumoto M, Kaji Y, Endo F, Nagashima H (2008) Production efficiency and telomere length of the cloned pigs following serial somatic cell nuclear transfer. J Reprod Dev 54(4):254–258CrossRefPubMedGoogle Scholar
  19. 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(5557):1089–1092CrossRefPubMedGoogle Scholar
  20. Lee K, Redel BK, Spate L, Teson J, Brown AN, Park KW, Walters E, Samuel M, Murphy CN, Prather RS (2013) Piglets produced from cloned blastocysts cultured in vitro with GM-CSF. Mol Reprod Dev 80(2):145–154CrossRefPubMedPubMedCentralGoogle Scholar
  21. Li L, Pang D, Chen L, Wang T, Nie D, Yan S, Ouyang H (2009) Establishment of a transgenic pig fetal fibroblast reporter cell line for monitoring Cre recombinase activity. DNA Cell Biol 28(6):303–308CrossRefPubMedGoogle Scholar
  22. Li J, Gao Y, Petkov S, Purup S, Hyttel P, Callesen H (2014) Passage number of porcine embryonic germ cells affects epigenetic status and blastocyst rate following somatic cell nuclear transfer. Anim Reprod Sci 147(1–2):39–46CrossRefPubMedGoogle Scholar
  23. Lillico SG, Proudfoot C, Carlson DF, Stverakova D, Neil C, Blain C, King TJ, Ritchie WA, Tan W, Mileham AJ, McLaren DG, Fahrenkrug SC, Whitelaw CB (2013) Live pigs produced from genome edited zygotes. Sci Rep 3:2847CrossRefPubMedGoogle Scholar
  24. Lillico SG, Proudfoot C, King TJ, Tan W, Zhang L, Mardjuki R, Paschon DE, Rebar EJ, Urnov FD, Mileham AJ, McLaren DG, Whitelaw CB (2016) Mammalian interspecies substitution of immune modulatory alleles by genome editing. Sci Rep 6:21645CrossRefPubMedPubMedCentralGoogle Scholar
  25. Matsuda T, Cepko CL (2007) Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A 104(3):1027–1032CrossRefPubMedPubMedCentralGoogle Scholar
  26. Meinke G, Bohm A, Hauber J, Pisabarro MT, Buchholz F (2016) Cre recombinase and other tyrosine recombinases. Chem Rev 116(20):12785–12820CrossRefPubMedGoogle Scholar
  27. Moon J, Kim S, Park H, Kang J, Park S, Koo O, da Torre BR, Saadeldin IM, Lee B, Jang G (2012) Production of porcine cloned embryos derived from cells conditionally expressing an exogenous gene using Cre–loxP. Zygote 20(4):423–425CrossRefPubMedGoogle Scholar
  28. Prather RS, Rowland RR, Ewen C, Trible B, Kerrigan M, Bawa B, Teson JM, Mao J, Lee K, Samuel MS, Whitworth KM, Murphy CN, Egen T, Green JA (2013) An intact sialoadhesin (Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcine reproductive and respiratory syndrome virus. J Virol 87(17):9538–9546CrossRefPubMedPubMedCentralGoogle Scholar
  29. 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(5):1228–1238CrossRefPubMedPubMedCentralGoogle Scholar
  30. 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):1571–1577CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ross JW, Fernandez de Castro JP, Zhao J, Samuel M, Walters E, Rios C, Bray-Ward P, Jones BW, Marc RE, Wang W, Zhou L, Noel JM, McCall MA, DeMarco PJ, Prather RS, Kaplan HJ (2012) Generation of an inbred miniature pig model of retinitis pigmentosa. Invest Ophthalmol Vis Sci 53(1):501–507CrossRefPubMedPubMedCentralGoogle Scholar
  32. Seraphin B, Simon M, Boulet A, Faye G (1989) Mitochondrial splicing requires a protein from a novel helicase family. Nature 337(6202):84–87CrossRefPubMedGoogle Scholar
  33. Song Y, Lai L, Li L, Huang Y, Wang A, Tang X, Pang D, Li Z, Ouyang H (2016) Germ cell-specific expression of Cre recombinase using the VASA promoter in the pig. FEBS open bio 6(1):50–55CrossRefPubMedGoogle Scholar
  34. Van Gorp H, Van Breedam W, Van Doorsselaere J, Delputte PL, Nauwynck HJ (2010) Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus. J Virol 84(6):3101–3105CrossRefPubMedGoogle Scholar
  35. Wells KD, Bardot R, Whitworth KM, Trible BR, Fang Y, Mileham A, Kerrigan MA, Samuel MS, Prather RS, Rowland RR (2017) Replacement of porcine CD163 scavenger receptor cysteine-rich domain 5 with a CD163-like homolog confers resistance of pigs to genotype 1 but not genotype 2 porcine reproductive and respiratory syndrome virus. J Virol 91(2):e01521–16CrossRefPubMedPubMedCentralGoogle Scholar
  36. Whitworth KM, Prather RS (2010) Somatic cell nuclear transfer efficiency: How can it be improved through nuclear remodeling and reprogramming? Mol Reprod Dev 77:1001–1015. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, Murphy SL, Samuel MS, Mao J, O’Gorman C, Walters EM, Murphy CN, Driver J, 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):78CrossRefPubMedPubMedCentralGoogle Scholar
  38. Whitworth KM, Rowland RR, Ewen CL, Trible BR, Kerrigan MA, Cino-Ozuna AG, Samuel MS, Lightner JE, McLaren DG, Mileham AJ, Wells KD, Prather RS (2016) Gene-edited pigs are protected from porcine reproductive and respiratory syndrome virus. Nat Biotechnol 34(1):20–22CrossRefPubMedGoogle Scholar
  39. Whitworth KM, Benne JA, Spate LD, Murphy SL, Samuel MS, Murphy CN, Richt JA, Walters E, Prather RS, Wells KD (2017) Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs. Transgenic Res 26(1):97–107CrossRefPubMedGoogle Scholar
  40. Xing X, Magnani L, Lee K, Wang C, Cabot RA, Machaty Z (2009) Gene expression and development of early pig embryos produced by serial nuclear transfer. Mol Reprod Dev 76(6):555–563CrossRefPubMedGoogle Scholar
  41. Yoshioka K, Suzuki C, Tanaka A, Anas IM, Iwamura S (2002) Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol Reprod 66(1):112–119CrossRefPubMedGoogle Scholar
  42. Yuan Y, Spate LD, Redel BK, Tian Y, Zhou J, Prather RS, Roberts RM (2017) Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation. Proc Natl Acad Sci U S A 114(29):E5796–E5804CrossRefPubMedPubMedCentralGoogle Scholar
  43. Zeyland J, Lipinski D, Slomski R (2014) The current state of xenotransplantation. J Appl Genet 56(2):211–218Google Scholar
  44. Zhao J, Whyte J, Prather RS (2010) Effect of epigenetic regulation during swine embryogenesis and on cloning by nuclear transfer. Cell Tissue Res 341(1):13–21CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Kristin M. Whitworth
    • 1
  • Raissa Cecil
    • 1
  • Joshua A. Benne
    • 1
  • Bethany K. Redel
    • 1
  • Lee D. Spate
    • 1
  • Melissa S. Samuel
    • 1
  • Randall S. Prather
    • 1
  • Kevin D. Wells
    • 1
  1. 1.Division of Animal SciencesUniversity of MissouriColumbiaUSA

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