The Research Of Myostatin Gene Knockout Somatic Cell Cloned Mongolian Sheep

In recent years, a negative regulatory factor-Myostatin (MSTN) had been discovered which can inhibit the growth of the skeletal muscle Myostatin after genetic mutations. This factor can lead to inactivation of gene function, then cause excessive development of animal muscle. The quantity and diameter of muscle fiber, the muscle mass increases sharply, the animals performanced for "double muscle" character. As for this, researchers got the animal models and gene knockout transgenic cloned animals through the muscle inhibition gene knockout, missing, mutation, gene modification in order to achieve the requirement in medicine and agriculture. For this study, the Mongolian sheep is one of the dominant species in Inner Mongolia autonomous region. It is of great significance of livestock breeding in autonomous region to cultivating good meat traits and high production Mongolian sheep.In this study,we selected30days of pregnancy Mongolian sheep fetus as material, then established the Mongolian sheep fetus fiber cell line successfully which was used in experiment of MSTN gene targeting through the organization block adherent method. At the same time,we took use of knockout technology to constructed MSTN gene exon3missing knockout vector of Mongolian sheep in vitro. We mainly built two vectors with a displacement type of screening marker and a TALEN knock out vector without selectie markers. The main results were as follows:1BGI sequencing company was commissioned for the sequencing of Mongolian sheep myostatin gene, after obtained the results of sequencing, comparison the result with the Ovies of MSTN gene sequences which on Genebank released for, primers were designed and we cloned Mongolian sheep myostatin gene successfully.2We designed primer, then amplified two homologous long arms of Mongolian sheep MSTN gene successfully by PCR, which sizes were5600bp and6000bp respectively, including all the exonl, intronl, exon2and intron2and part of the promoter and a small number of exon3,homologous short arm is1100bp. including some exon3and the3’translation section sequence.then we connected the long and homologous arm respectively with the skeleton vector pPNT Ⅲ and pEGF-N1which had positive and negative selection marker genes.We constructed displacement type targeting vector of MSTN gene exon3lost successfully by TA cloning and seamless cloning method. The identification of enzyme digestion and sequencing icdicatcd that both displacement type carrier had been built correctly.3We constructed two TALEN vectors (197and198) which against to17base mutation in exon3of Mongolian sheep muscle inhibition gene without screening markers and one vector(199) of the17base mutations in exon2. Only two TALEN vectors (197and198) are verified to have knockout activity through the active identification.4We established Mongolia sheep fetal Fibroblasts through organization block adherent method and the the cell lines grow well. The replacement vector and TALEN vectors were transfected with the use of electricity and by mediating of LipofectamineTM2000liposome transfection Mongolian sheep fetal fibroblasts. We use the methods of drug of G418and the infinite dilution mouth pipette to screening the monoclonal cell. Part of cell lines were used to identify whether the gene had been knockout, part of which were cryopreserved. After this, we extracted DNA of screened cell lines, PCR, and identification. The final identification of the correct20of TALEN transgenic cell lines were done PCR again, and connected the PCR products (including targeted points in the third exon) into the pMD-19, after colony PCR, we set15samples of single clone picked randomly to sequence for the purpose of identifying whether it had bases mutations in the targeted point. The sequencing result proved that there are about four cell lines of the bases lost in MSTN gene of197vector.and single-base mutation or missing occured in only three cell lines of198vector.5We selected four cell lines P66, E20, P42and P54(which were lacked19,11.8and11bases in the vicinity of the target site) as the donor cells,and mature oocytes without nuclear as receptor cclls,then built reconstructed gene knockout embryos through microscopic injections and performed embryo transfer.As the Results, there were61receptor sheep had been transplanted in total for four different kinds of cell lines. By B-test, there are a total of20pregnant sheep.6The knockout lambs has been born11. There are four lambs has been detected, two double knockout allele Lamb (base deletion19bp).2lambs are single allele knockout.7lambs are identifing, and2receptor to be produced.