Generation Of Porcine MSTN Double-knockout Cell Model By Genome Modification

Myostatin(MSTN)gene encodes myostatin protein,negatively regulation of muscle growth.MSTN knockout domestic animal,their muscle phenotype is "double muscle type",which used to be applied to improve the meat quality of domestic animals.Its pharmacological effects with medical value,is expected to apply in the treatment of muscle atrophy,especially the potential for the treatment of cardiac contraction function.Another study showed that inhibition of the expression of animal MSTN is able to improve glucose intake ability by enhancing animal insulin sensitivity,which may be applied to treat type II diabetes;inhibition of the expression of MSTN leads to decrease the fat contain in animal blood relatively,which may be applied to improve the dyslipidemia and atherosclerosis effectively.MSTN knockout cell model can be used for the research and development of new drugs associated diseases and the further research of MSTN function.In this study,marker gene was inserted into target site by homologous recombination mediated by CRISPR/Cas9 to inactivate first MSTN allele,and to screen cell clones by gene markers,and completion of the first shooting;Cre/Lox P system to delete marker genes,deletion of the positive marker gene,completion of the deletion of marker genes;marker gene was inserted into target site by homologous recombination mediated by CRISPR/Cas9 to inactivate second MSTN allele,and to screen cell clones by gene markers,and completion of the second shooting.After cell transfection,G418 resistance screening,fluorescence microscopy and flow cytometry were used to screen and identify the fluorescent protein markers;cells were verified by PCRproduct gel electrophoresis,PCR product sequencing,Southern blot and Western blot.Comprehensive analysis of the experimental results showed that: the first shooting test,generation of the labeled MSTN gene knockout cell line PK3108;deletion mark gene test,generation of gene knockout cell lines PK3108-1,PK3108-2,PK3108-3 with marker-free;the second shooting test,generation of MSTN bi-allelic knockout cell line L18 with positive markers.In this study,we successfully constructed a porcine MSTN double allelic knockout cell model by using CRISPR/Cas9 and Cre/Lox P techniques,which provided a reference to double allelic knockout in vitro.In this study,MSTN single-and double-allelic cell lines were constructed,which provided cell models for the development of new drugs associated with diseases and the further study of MSTN function.In summary,this study provided a new reference for construction of biallelic knockout cell model in vitro and was good for the development of pig breeding and researches on skeletal muscle and myocardial pathology,and treatment of type II diabetes and improving dyslipidemia and atherosclerosis.