Production of MSTN mutated Korean beef cow
The aim of this study was to produce MSTN mutated Korean beef cattle. Microinjection was performed on fertilized embryos under RNA2X conditions and cultured blastocysts were used for embryo transfer. The experiment was repeated four times and 595 oocytes were used to generate mutant embryos. The cleavage rate was 59.0 ± 21.0% and 86 blastocysts (14.5 ± 14.0%) were produced. A total of 26 blastocysts with high quality morphology were transferred to surrogate mothers (one blastocyst per recipient). Some of leftover blastocyst (n = 28) were used to assess the mutation occurrence on MSNT and it was determined to be 71.6 ± 44.3%.
After embryo transfer, 19 of the 26 surrogates were pregnant and 2 of the 19 fetuses were absorbed in the middle of pregnancy. A total of 17 calves (I.D.: #1 ~ #17) were live-born and one calf has a stillbirth (#14) from dystocia. Deep sequencing analysis showed mutations in 3 out of the 17 animals (Fig. 2A). Their MSTNmutation rates were 10.5, 45.4, and 99.9% for #6, #14, and #17, respectively. In addition, MSTN mutation was detected in #17 by T7E1 assay and there were no off-target effects (Fig. 2C). Real-time PCR was conducted to assess the MSTN RNA level of primary cells from #17 and #14. In both individuals the level of MSTN RNA was significantly decreased with wild type (Fig. 2D). Especially in #17 with the -12 deletion, enhanced muscle growth was observed compared with wild type calf (Fig. 2E). There were no abnormal values in a blood test performed to evaluate the general health status of 5 offspring (#4, #6, #7, #9 and #17) including the knockout one (#17) at 8-months-old (Table 1).