Research On Mstn Of Mutated Rabbits By CRISPR/Cas9

Myostatin?Myostatin,MSTN?is a negative regulator of skeletal muscle growth.Researches have shown that natural mutations in the MSTN gene can cause double-muscle buttock traits in rats,cattle,pigs,and sheep,improving animal feed conversion efficiency and meat production.We carried out rabbit MSTN gene editing research,with a view to obtaining MSTN gene editing rabbitsuses by CRISPR/Cas9 system,improving rabbit growth speed,and cultivating a new"double muscle buttocks"rabbit strain.The paper obtained the following research results:?1?Rabbit MSTN g RNA designed and vector construction:A total of8 g RNAs were designed for exon 1 and exon 3 of rabbit MSTN by software,and 8 g RNAs expression vectors were constructed by ligation and directional cloning.Liposome transfection was used to treat 293T cells,and the ratio of red fluorescence to green fluorescence was observed by flow cytometry to determine the exogenous knockout efficiency of 8 g RNAs designed by the CRISPR/Cas9 system.The results showed that The designed four g RNA in exon3 knockout efficiency is significantly higher than the four g RNA on exon 1,the top three knockout efficiency reached 90.7%,89.5%and 77.7%,respectively.?2?Gene editing efficiency of rabbit fetal fibroblasts:The primary fibroblasts of rabbits were cultured by tissue block adherence method,and the transfection of the fibroblasts in the logarithmic growth phase was carried out by electrical transfection.The editing efficiency of 4 target sequences g5-g8 on exon 3 is 25%,45%,30%and 40%,respectively.The sequencing results show that the g RNA with the highest editing efficiency is g6 and g8.?3?Preparation and detection of rabbit MSTN knockout model:Collect zygotes of female rabbits,mix g6 and g8 transcribed in vitro with Cas9at a ratio of 1:5,and inject exogenous RNA into zygotes by microinjection,A total of 30 embryoes transfer recipient female rabbits,the experimental pregnancy rate was 33.33%?10/30?,the survival rate of pups was 61.11%?23/36?,and the positive rate among surviving pups was 21.74%?5/23?.Further gene sequencing tests showed that the five gene knockout rabbits of F0generation were all chimeras,three of which were MSTN^+/-male rabbits with normal reproductive ability,and two MSTN^-/-female rabbits had been bred many times and had been unable to become pregnant.The F1 progeny rabbits obtained from MSTN^+/-male rabbits detected the same genotype as the F0 progeny.?4?Observation of the rabbits MSTN knockout model:some premature fetuses of MSTN-KO rabbits exhibited"big tongue syndrome",and some of the live rabbits born also had"big tongue syndrome"in MSTN-KO rabbits,which caused breastfeeding Obstacle,died 2-3 days after birth.At 180days of age,MSTN-KO F0 rabbits showed a"double muscle buttocks"phenomenon,MSTN-KO rabbits 3708±43.06g,WT rabbits 3224±48.64g,the difference was very significant?P<0.001,N=5?,Compared with WT rabbits,the average weight gain of MSTN-KO rabbits is 484g.Western Blot results show that MSTN-KO affects the expression of MSTN genes.The results of tissue section analysis showed that the MSTN-KO rabbit had a larger cross-sectional area of hip muscle fibers than the normal control group.Conclusion:In this experiment,CRISPR/Cas9 technology was used to successfully obtain MSTN-KO rabbits.Large tongue syndrome was found in dead and some infertile rabbits,normal MSTN-KO rabbits showed For the"double muscle buttocks"feature,This research laid the foundation for breeding new strains of double-muscle hip rabbits with fast growth rate and high meat production rate.