Disruption Of Myostatin Gene In Pigs By Engineered Nucleases

Background:Myostatin, or growth and differentiation factor8(GDF8), is a highly conserved member of the TGF-β superfamily and possesses all of the structural components common to the family, and it was proved to be negative regulatory factor to the growth of skeletal muscle. In mammals, the mutant of myostatin by natural or artificial induced the increase of skeletal muscle number that made a new path to improve the yield of livestock. The homology targeting, RNAi and engineered nucleases(ENs), the efficiency of the first method is very low; the second introduced transgene to genome of animals; while the engineered nucleases not only the efficiency is higher than homology targeting, but also not introduce any transgene to genome of animals. EN is consists of DNA binding protein which recognizes the specific DNA sequence and nucleases without specificity. The ENs could bind to the specific DNA sequence and make double strand break (DSB), the DSB is repaired by the two repair mechanism---Homology-directed repair (HDR) and Non-homologous end joining (NHEJ) in vivo. The genetic information lost when repair the DSB, then the target gene destructed.Methods and Results:In our research, we designed two different ENs that both target the first exon of myostatin gene of pig, the effect and activity of the ENs are identified in the porcine oocyte and PEF cells.(1) Transfect the plasmid of ZFNs and TALENs to PEF cells respectively, amplify the myostatin gene from PEF cell genome after48h’s or72h’s culture, then analysis the PCR fragments using T7EI assay and DNA sequence analysis. Both of the ZFNs and TALENs are effect in PEF cell, they knockout the myostatin gene from the porcine genome successfully, and the efficiency of TALENs (11.1%) is higher than ZFNs (4.8%).(2) Transcript the ZFNs and TALENs into mRNA in vitro, and perform the mRNA microinject to porcine oocytes. As the porcine oocytes are very sensitive to the change of temperature, the blastula ratio of injected embryos is difference in different seasons (p<0.05). Compared to the control, the blastula ratio of the injected embryos is not significant difference (p>0.05), the blastitula ratio of embryos are higher in summer than in winter; it indicates that neither the microinjection program nor the mRNA encoding ZFNs protein effects the development of the embryos. The injected embryos that development to blastula are collected for the detection using PCR, analysis the PCR fragments using T7EI assay and DNA sequence analysis. Both of the ZFNs and TALENs are also effect in porcine oocytes, and the efficiency of the ZFNs (5.31%) and TALENs (11.3%) are similar with the results in PEF cells, too. However, the weight of mutant cell in one embryo that developed to blastula is lower in TALENs treated embryos than ZFNs.Conclusions:In our research, both of the ZFNs and TALENs are knockout the myostatin gene from porcine genome successfully in either PEF cells or porcine embryos, and the knockout efficiency of TALENs is higher than ZFNs. All of these provided the basis of the transgenic pig which is deficiency in myostatin gene, it could be heavy producing and no harm to environment.