The Study On The Modification Of MSTN Gene In Pigs By CRISPR/Cas9 System

CRISPR is a special DNA sequence repeats family,is a specific immune defense mechanisms of bacteria and archaea resulting in constant evolution.In the vicinity of the CRISPR locus there is a family known as the Cas protein that acts as a function of coding nuclease,helicase,polymerase,and ribonucleic acid.The CRISPR / Cas9 system consists essentially of repeats,the protospacers,and the Cas protein family upstream of the repeat sequence?CRISPR / Cas9 system mainly has I,II,III three types,II type CRISPR / Cas9 system because of its simple structure,much favored by researchers and transformed into mammalian genomic DNA targeted modification of the tool,the system The single-stranded RNA(sgRNA)and Cas9 protein of the dimer structure were only required for the cleavage of the target sequence.The gRNA identified the target gene sequence containing PAM(NGG)by directing Cas9 to double-strand the target gene DNA Source recombination mediated by gene knockout is possible.Myostatin(myostatin)is a secretory polypeptide that belongs to the TGF-? superfamily member and has a biological structure shared by the family.MSTN gene is a negative regulator of skeletal muscle,mainly by regulating the proliferation of mammalian myoblasts affect muscle growth and development.Biological changes in the natural state and the use of gene knockout technology to knock out the MSTN gene animal muscle volume will increase sharply,resulting in double muscle traits,we live in contact with the Belgian blue cattle and Piedmont beef biceps The trait belongs to the MSTN gene mutation.MSTN gene mutation "double muscle traits" can greatly improve the status of the world's livestock industry,to improve the lean meat rate of livestock,to speed up the slaughter cycle is of great significance.At the same time,in the biomedical industry,the use of MSTN mutations,for the treatment of certain muscle atrophy,muscle dysplasia provides the possibility.At the same time,the study found that MSTN gene can inhibit the formation of fat,which for the treatment of obesity and diabetes has a very important significance.Pork as China's major sources of meat supply,and people's lives are closely linked,and the pig in the physiological,organizational structure,anatomical analysis,nutrition and other aspects of human and very similar,very suitable for animal disease model to study human disease.Therefore,it is very important to produce MSTN gene mutant pigs for the breeding of livestock breeding,to speed up the growth cycle of pigs and to prepare model animals.In this study,we selected the T1 target site of the first exon on the MSTN gene and the T2 target site of the third exon as the target.The HRX-2MCS vector possessed the green fluorescent protein gene and the G418 resistance gene as the positive screening,and the red fluorescent protein gene was used as negative screening in the HRX-2MCS vector as the backbone vector.The homologous left and right arms of the target sequence were cloned into the vector via the multiple cloning sites on the HRX-2MCS vector to prepare the donor vectors dT1 and dT2 and the vector T11 of the Cas9 protease system containing the T1 and T2 target sites was prepared by the company And T21.(DT1 and T11,dT2 and T21)were transfected into PK-15 cell line by electroporation method.The positive effects of G418 and green fluorescent protein and the negative screening of red fluorescent protein were used to select the single Clonal cell lines.The genomic DNA of the monoclonal cell line was extracted and the clonal spots were detected by PCR and sequencing.The positive clonal cell lines were 29 and the T2 positive locus was 26.In summary,this study confirmed that the use of CRISPR / Cas9 gene knockout technology to prepare MSTN gene mutations in animal models there is a possibility.