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Transgenic Efficiency of FoxN1-targeted Pig Parthenogenetic Embryos
여재훈,황인설,박재경,임석기,박응우,이정웅,박춘근,황성수,권대진 사단법인 한국동물생명공학회 2014 한국동물생명공학회지 Vol.29 No.4
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.
Hh-Ag1.5 처리가 돼지 체외수정란의 발육 및 세포사멸에 미치는 영향
권대진,여재훈,노원근,곽태욱,오건봉,옥선아,임석기,박진기,황성수 韓國受精卵移植學會 2013 한국동물생명공학회지 Vol.28 No.3
The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.