Reproductive technologies needed for the generation of precise gene-edited pigs in the pathways from laboratory to farm

  • Ching-Fu Tu (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Shu-Hui Peng (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Chin-kai Chuang (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Chi-Hong Wong (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute) ;
  • Tien-Shuh Yang (Division of Animal Technology, Animal Technology Research Center, Agricultural Technology Research Institute)
  • Received : 2022.10.11
  • Accepted : 2022.11.07
  • Published : 2023.02.01


Gene editing (GE) offers a new breeding technique (NBT) of sustainable value to animal agriculture. There are 3 GE working sites covering 5 feasible pathways to generate GE pigs along with the crucial intervals of GE/genotyping, microinjection/electroporation, induced pluripotent stem cells, somatic cell nuclear transfer, cryopreservation, and nonsurgical embryo transfer. The extension of NBT in the new era of pig breeding depends on the synergistic effect of GE and reproductive biotechnologies; the outcome relies not only on scientific due diligence and operational excellence but also on the feasibility of application on farms to improve sustainability.



This article was financially supported (NSTC 110-2313-B-866-002) by the National Science and Technology Council, Executive Yung, Taiwan.


  1. Tu CF, Chuang CK, Yang TS. The application of new breeding technology based on gene editing in pig industry. Anim Biosci 2022;35:791-803.
  2. Tanihara F, Hirata M, Otoi T. Current status of the application of gene editing in pigs. J Reprod Dev 2021;67:177-87.
  3. Zhang J, Khazalwa EM, Abkallo HM, et al. The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research. J Genet Genomics 2021;48:347-60.
  4. Betermier M, Bertrand P, Lopez BS. Is non-homologous end-joining really an inherently error-prone process? PLoS Genet 2014;10:e1004086.
  5. Bennett EP, Petersen BL, Johansen IE, et al. INDEL detection, the 'Achilles heel' of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels. Nucleic Acids Res 2020;48:11958-81.
  6. Chuang CK, Lin WM. Points of view on the tools for genome/gene editing. Int J Mol Sci 2021;22:9872.
  7. Chuang CK, Lee KH, Fan CT, Su YS. Porcine type III RNA polymerase III promoters for short hairpin RNA expression. Anim Biotechnol 2009;20:34-9.
  8. Christou-Kent M, Dhellemmes M, Lambert E, Ray PF, Arnoult C. Diversity of RNA-binding proteins modulating posttranscriptional regulation of protein expression in the maturing mammalian oocyte. Cells 2020;9:662.
  9. Li L, Zheng P, Dean J. Maternal control of early mouse development. Development 2010;137:859-70.
  10. Chuang CK, Chen CH, Huang CL, et al. Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors. Anim Biotechnol 2017;28:174-81.
  11. Tu CF, Chuang CK, Hsiao KH, et al. Lessening of porcine epidemic diarrhoea virus susceptibility in piglets after editing of the CMP-N-glycolylneuraminic acid hydroxylase gene with CRISPR/Cas9 to nullify N-glycolylneuraminic acid expression. PLoS One 2019;14:e0217236.
  12. Hung SW, Chuang Ck. Wong CH, et al. Activated macrophages of CD 163 gene edited pigs generated by direct cytoplasmic microinjection with CRISPR gRNA/Cas9 mRNA are resistant  to PRRS virus assault. Anim Biotechnol 2022;May 4:1-14.
  13. Tanihara F, Hirata M, Nguyen NT, et al. Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation. Anim Sci J 2019;90:55-61.
  14. Tanihara F, Hirata M, Nguyen NT, et al. Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/ Cas9 system into in vitro-fertilized zygotes. BMC Biotechnol 2020;20:40.
  15. Tanihara F, Hirata M, Nguyen NT, et al. Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes. Anim Biotechnol 2021;32:147-54.
  16. Ren C, Xu K, Segal DJ, Zhang Z. Strategies for the enrichment and selection of genetically modified cells. Trends Biotechnol 2019;37:56-71.
  17. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663-76.
  18. Esteban MA, Xu J, Yang J, et al. Generation of induced pluripotent stem cell lines from Tibetan miniature pig. J Biol Chem 2009;284:17634-40.
  19. Ezashi T, Telugu BP, Alexenko AP, et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci USA 2009;106:10993-8.
  20. Wu Z, Chen J, Ren J, et al. Generation of pig induced pluripotent stem cells with a drug-inducible system. J Mol Cell Biol 2009;1:46-54.
  21. West FD, Terlouw SL, Kwon DJ, et al. Porcine induced pluripotent stem cells produce chimeric offspring. Stem Cells Dev 2010;19:1211-20.
  22. West FD, Uhl EW, Liu Y, et al. Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs. Stem Cells 2011;29:1640-3.
  23. Liu K, Ji G, Mao J, et al. Generation of porcine-induced pluripotent stem cells by using OCT4 and KLF4 porcine factors. Cell Reprogram 2012;14:505-13.
  24. Du X, Feng T, Yu D, et al. Barriers for deriving transgene-free pig iPS cells with episomal vectors. Stem Cells 2015;33:3228-38.
  25. Chen CH, Su YH, Lee KH, Chuang CK. Germline competent pluripotent mouse stem cells generated by plasmid vectors. Anim Biotechnol 2016;27:157-65.
  26. Kim JY, Nam Y, Rim YA, Ju JH. Review of the current trends in clinical trials involving induced pluripotent stem cells. Stem Cell Rev Rep 2022;18:142-54.
  27. Howden SE, Maufort JP, Duffin BM, Elefanty AG, Stanley EG, Thomson JA. Simultaneous reprogramming and gene correction of patient fibroblasts. Stem Cell Rep 2015;5:1109-18.
  28. Cheng D, Guo Y, Li Z, et al. Porcine induced pluripotent stem cells require LIF and maintain their developmental potential in early stage of embryos. PLoS One 2012;7:e51778.
  29. Yuan Y, Lee K, Park KW, et al. Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos. Cell Cycle 2014;13:1265-76.
  30. Kim E, Hwang SU, Yoo H, et al. Putative embryonic stem cells derived from porcine cloned blastocysts using induced pluripotent stem cells as donors. Theriogenology 2016;85: 601-16.
  31. Fan N, Chen J, Shang Z, et al. Piglets cloned from induced pluripotent stem cells. Cell Res 2013;23:162-6.
  32. Yu D, Wang J, Zou H, et al. Silencing of retrotransposonderived imprinted gene RTL1 is the main cause for postimplantational failures in mammalian cloning. Proc Natl Acad Sci 2018;115:E11071-80.
  33. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810-3.
  34. Polejaeva IA, Chen SH, Vaught TD, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000;407:86-90.
  35. Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems. Nat Biotechnol 2000;18:1055-9.
  36. Onishi A, Iwamoto M, Akita T, et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science 2000;289:1188-90.
  37. Hua Z, Xu G, Liu X, et al. Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig. Pol J Vet Sci 2016;19:205-12.
  38. Richter A, Kurome M, Kessler B, et al. Potential of primary kidney cells for somatic cell nuclear transfer mediated transgenesis in pig. BMC Biotechnol 2012;12:84.
  39. Fahrudin M, Kikuchi K, Kurniani Karja NW, et al. Development to the blastocyst stage of porcine somatic cell nuclear transfer embryos reconstructed by the fusion of cumulus cells and cytoplasts prepared by gradient centrifugation. Cloning Stem Cells 2007;9:216-28.
  40. Li X, Zhang P, Jiang S, et al. Aging adult porcine fibroblasts can support nuclear transfer and transcription factor-mediated reprogramming. Anim Sci J 2018;89:289-297.
  41. Li Z, He X, Chen L, et al. Bone marrow mesenchymal stem cells are an attractive donor cell type for production of cloned pigs as well as genetically modified cloned pigs by somatic cell nuclear transfer. Cell Reprogram 2013;15:459-70.
  42. Park CH, Jeoung YH, Uh KJ, et al. Extraembryonic endoderm (XEN) cells capable of contributing to embryonic chimeras established from pig embryos. Stem Cell Rep 2021;16:212-223.
  43. Dang-Nguyen TQ, Wells D, Haraguchi S, et al. Combined refinements to somatic cell nuclear transfer methods improve porcine embryo development. J Reprod Dev 2020;66:281-6.
  44. Jiao D, Cheng W, Zhang X, et al. Improving porcine SCNT efficiency by selecting donor cells size. Cell Cycle 2021;20: 2264-77.
  45. Yin XJ, Tani T, Yonemura I, et al. Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes. Biol Reprod 2002;67:442-6.
  46. Du Y, Kragh PM, Zhang Y, et al. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 2007;68:1104-10.
  47. Betthauser J, Forsberg E, Augenstein M, et al. Production of cloned pigs from in vitro systems. Nat Biotechnol 2000;18:1055-9.
  48. Du Y, Kragh PM, Zhang Y, et al. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 2007;68:1104-10.
  49. de Macedo MP, Glanzner WG, Rissi VB, et al. A fast and reliable protocol for activation of porcine oocytes. Theriogenology 2019;123:22-9.
  50. Lee K, Davis A, Zhang L, et al. Pig oocyte activation using a Zn2+ chelator, TPEN. Theriogenology 2015;84:1024-32.
  51. Wang X, Qu J, Li J, et al. Epigenetic reprogramming during somatic cell nuclear transfer: recent progress and future directions. Front Genet 2020;11:205.
  52. Zeng Y, Chen T. DNA methylation reprogramming during mammalian development. Genes 2019;10:257.
  53. Ju S, Rui R, Lu Q, Lin P, Guo H. Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro. J Assist Reprod Genet 2010;27:49-59.
  54. Park HB, Park YR, Kim MJ, Jung BD, Park CK, Cheong HT. Endoplasmic reticulum (ER) stress inhibitor or antioxidant treatments during micromanipulation can inhibit both ER and oxidative stresses in porcine SCNT embryos. Dev Reprod 2020;24:31-41.
  55. Park J, Lai L, Samuel MS, et al. Disruption of mitochondrionto-nucleus interaction in deceased cloned piglets. PLoS One 2015;10:e0129378.
  56. Srirattana K, Kaneda M, Parnpai R. Strategies to improve the efficiency of somatic cell nuclear transfer. Int J Mol Sci 2022;23:1969.
  57. Chen Z, Zhang Y. Role of mammalian DNA methyltransferases in development. Annu Rev Biochem 2019;89:135-58.
  58. Gu TP, Guo F, Yang H, et al. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 2011;477:606-10.
  59. Inoue A, Zhang Y. Replication-dependent loss of 5-hydroxymethylcytosine in mouse preimplantation embryos. Science 2011;334:194.
  60. Huang Y, Tang X, Xie W, et al. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos. Biochem Biophys Res Commun 2011;411:397-401.
  61. Kere M, Siriboon C, Lo NW, Nguyen NT, Ju JC. Ascorbic acid improves the developmental competence of porcine oocytes after parthenogenetic activation and somatic cell nuclear transplantation. J Reprod Dev 2013;59:78-84.
  62. Yin R, Mao SQ, Zhao B, et al. Ascorbic acid enhances Tetmediated 5-methylcytosine oxidation and promotes DNA demethylation in mammals. J Am Chem Soc 2013;135:10396-403.
  63. Zhao M, Hur TY, No J, et al. Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa). Asian-Australas J Anim Sci 2017;30:944-9.
  64. Lee K, Hamm J, Whitworth K, et al. Dynamics of TET family expression in porcine preimplantation embryos is related to zygotic genome activation and required for the maintenance of NANOG. Dev Biol 2014;386:86-95.
  65. Guo Z, Lv L, Liu D, Fu B. Effects of trichostatin A on pig SCNT blastocyst formation rate and cell number: A metaanalysis. Res Vet Sci 2018;117:161-6.
  66. Zhao J, Ross JW, Hao Y, et al. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biol Reprod 2009;81:525-30.
  67. Zhao J, Hao Y, Ross JW, et al. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram 2010;12:75-83.
  68. Liang S, Zhao MH, Choi JW, et al. Scriptaid treatment decreases DNA methyltransferase 1 expression by induction of microRNA-152 expression in porcine somatic cell nuclear transfer embryos. PLoS One 2015;10:e0134567.
  69. Jeong PS, Sim BW, Park SH, et al. Chaetocin improves pig cloning efficiency by enhancing epigenetic reprogramming and autophagic activity. Int J Mol Sci 2020;21:4836.
  70. Kobayashi W, Kurumizaka H. Structural transition of the nucleosome during chromatin remodeling and transcription. Curr Opin Struct Biol 2019;59:107-14.
  71. Antony J, Oback F, Chamley LW, et al. Transient JMJD2Bmediated reduction of H3K9me3 levels improves reprogramming of embryonic stem cells into cloned embryos. Mol Cell Biol 2013;33:974-83.
  72. Matoba S, Liu Y, Lu F, et al. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell 2014;159:884-95.
  73. Chung YG, Matoba S, Liu Y, et al. Histone demethylase expression enhances human somatic cell nuclear transfer effciency and promotes derivation of pluripotent stem cells. Cell Stem Cell 2015;17:758-66.
  74. Liu Z, Cai Y, Wang Y, et al. Cloning of macaque monkeys by somatic cell nuclear transfer. Cell 2018;172:881-7.e7.
  75. Weng XG, Cai MM, Zhang YT, et al. Improvement in the in vitro development of cloned pig embryos after kdm4a overexpression and an H3K9me3 methyltransferase inhibitor treatment. Theriogenology 2020;146:162-70.
  76. Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9. Nat Chem Biol 2005;1:143-5.
  77. Jeong PS, Sim BW, Park SH, et al. Chaetocin improves pig cloning efficiency by enhancing epigenetic reprogramming and autophagic activity. Int J Mol Sci 2020;21:4836.
  78. Jeong PS, Yang HJ, Park SH, et al. Combined chaetocin/trichostatin A treatment improves the epigenetic modification and developmental competence of porcine somatic cell nuclear transfer embryos. Front Cell Dev Biol 2021;9:709574.
  79. Yoshino J, Kojima T, Shimizu M, Tomizuka T. Cryopreservation of porcine blastocysts by vitrification. Cryobiology 1993;30:413-22.
  80. Katayama KP, Stehlik J, Kuwayama M, Kato O, Stehlik Ed. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003;80:223-4.
  81. Esaki R, Ueda H, Kurome M, et al. Cryopreservation of porcine embryos derived from in vitro-matured oocytes. Biol Reprod 2004;71:432-7.
  82. Cuello C, Gil MA, Parrilla I, et al. Vitrification of porcine embryos at various developmental stages using different ultra-rapid cooling procedures. Theriogenology 2004;62: 353-61.
  83. Cuello C, Sanchez-Osorio J, Alminana C, et al. Effect of the cryoprotectant concentration on the in vitro embryo development and cell proliferation of OPS-vitrified porcine blastocysts. Cryobiology 2008;56:189-94.
  84. Du Y, Li J, Kragh PM, et al. Piglets born from vitrified cloned blastocysts produced with a simplified method of delipation and nuclear transfer. Cloning Stem Cells 2007;9:469-76.
  85. Nakano K, Matsunari H, Nakayama N, et al. Cloned porcine embryos can maintain developmental ability after cryopreservation at the morula stage. J Reprod Dev 2011;57:312-6.
  86. Jia B, Xiang D, Guo J, et al. Successful vitrification of earlystage porcine cloned embryos. Cryobiology 2020;97:53-9.
  87. Du X, Zhuan Q , Cheng K, et al. Cryopreservation of porcine embryos: recent updates and progress. Biopreserv Biobank 2021;19:210-8.
  88. Kamoshita M, Kato T, Fujiwara K, et al. Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated epsilon-poly-L-lysine. PLoS One 2017;12:e0176711.
  89. Li J, Rieke A, Day BN, Prather RS. Technical note: porcine non-surgical embryo transfer. J Anim Sci 1996;74:2263-8.
  90. Suzuki C, Iwamura S, Yoshioka K. Birth of piglets through the non-surgical transfer of blastocysts produced in vitro. J Reprod Dev 2004;50:487-91.
  91. Ducro-Steverink DW, Peters CG, Maters CC, Hazeleger W, Merks JWM. Reproduction results and offspring performance after non-surgical embryo transfer in pigs. Theriogenology 2004;62:522-31.
  92. Cuello C, Berthelot F, Martinat-Botte F, et al. Piglets born after non-surgical deep intrauterine transfer of vitrified blastocysts in gilts. Anim Reprod Sci 2005;85:275-86.
  93. Yoshioka K, Noguchi M, Suzuki C. Production of piglets from in vitro-produced embryos following non-surgical transfer. Anim Reprod Sci 2012;131:23-9.
  94. Gomis J, Cuello C, Sanchez-Osorio J, et al. Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos-evaluation of critical steps of the procedure. Theriogenology 2012;78:1339-49.
  95. Martinez EA, Nohalez1 A, Martinez CA, et al. The recipients' parity does not influence their reproductive performance following non-surgical deep uterine porcine embryo transfer. Reprod Dom Anim 2016;51:123-9.
  96. Angel MA, Gil MA, Cuello C, et al. An earlier uterine environment favors the in vivo development of fresh pig morulae and blastocysts transferred by a nonsurgical deep-uterine method. J Reprod Dev 2014;60:371-6.
  97. Tajima S, Uchikura K, Kurita T, et al. Insemination of recipient sows improves the survival to term of vitrified and warmed porcine expanded blastocysts transferred non-surgically. Anim Sci J 2020;91:e13453.
  98. Hirayama Y, Takishita R, Misawa H, et al. Non-surgical transfer of vitrified porcine embryos using a catheter designed for a proximal site of the uterus. Anim Sci J 2020;91:e13457.
  99. Tajima S, Motoyama S, Wakiya Y, et al. Piglet production by non-surgical transfer of vitrified embryos, transported to commercial swine farms and warmed on site. Anim Sci J 2020;91:e13476.