Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Transgenic Efficiency of FoxN1-targeted Pig Parthenogenetic Embryos

  • Yeo, Jae-Hoon (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Hwang, In-Sul (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Park, Jae Kyung (Department of Animal Biotechnology, Kangwon National University) ;
  • Kwon, Dae-Jin (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Im, Seoki (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Park, Eung-Woo (Animal Biotechnology Division, National Institute of Animal Science, RDA) ;
  • Lee, Jeong-Woong (Department of Animal Biotechnology, Kangwon National University) ;
  • Park, Choon-Keun (Research Center of Integrative Cellulomics, Korea Research Institute of Bioscience and Biotechnology) ;
  • Hwang, Seongsoo (Animal Biotechnology Division, National Institute of Animal Science, RDA)
  • Received : 2014.12.09
  • Accepted : 2014.12.21
  • Published : 2014.12.31
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Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

Keywords

References

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