Transgenic Efficiency of FoxN1-targeted Pig Parthenogenetic Embryos

Abstract

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