earticle

영어

Transgenic animals has been produced by using microinjection method for target gene editing. However, the producing efficiency of gene edited embryos in livestock is very low. Therefore, the objective of this study is production of high quality blastocyst and establishment of appropriate microinjection condition for gene editing embryos. Frist, we treated the cytochalasin B (CB) within injection medium for reduction of microinjection damage, and microinjected embryos were cultured in CR1aa medium supplemented with 0.1 μM melatonin. As a result, blastocysts development of microinjected embryos in CB treated group was significantly higher than that of non-treated group (p<0.05, CB+, 18.5±2.0% vs CB-, 11.2±3.5%). After microinjection under CB treated condition, blastocyst formation was significantly increased (p<0.05) in Mela treated group. Next, to investigate DNA damage in blastocyst stage embryos, fluorescence expressions of H2AX139ph (DNA damage protein) and RAD51 (DNA repairs protein) were observed by using immunofluorescence staining. DNA damage in developing blastocysts was reduced in the CB and Mela treated embryos. Finally, we identified the blastocyst development after microinjection by using CRISPR/Cas9 system according to protein or mRNA of Cas9. Green fluorescence expressions by GFP were increased in developing blastocysts derived from Cas9 protein microinjected embryos. Moreover, gene targeting rate in GFP expressed blastocysts was increased in Cas9 protein injected group (p<0.05; Cas9 protein, 30% vs Cas9 mRNA, 20%). These results demonstrated that positive effect of CB and Mela treatment improves the blastocysts qualities and their developmental competence via reduction of microinjection damage in gene editing bovine embryos. We also verified the improvement of knock-in efficiency in microinjected bovine embryos by using Cas9 protein.