Establishment Of Label-Freed CRISPR/Cas9-edited MSTN Cell Strains In Mongolia Bovine

Myostatin(MSTN)is a member of the TGF-?(transforming growth factor beta)superfamily.which is a secreted glycoprotein and a negative regulator of muscle development.It is expressed mainly in skeletal muscle.It has been recently found that overexpression of the gene may cause muscle atrophy and the gene mutation can cause gene inactivation,then leading to increase of animal muscle fiber number and diameter,a significant character of "double muscle".Mongolian cattle is an Inner Mongolia plateau characteristics species with the low meat production rate.Therefore,it makes sense to produce Mongolian cattle with good properties and high meat production rate for protect the gennplasm resources,breeding work.It has a reference value and significance for treating diseases about muscle development by gene editing.This study aimed to design and construct a safety and label-free carriers containing a MSTN editing by CRISPR/Cas9,and established an Mongolian cattle fetal skin fibroblastcell line of 45 days pregnancy by tissue explants adherent method.We design and construct three CRISPR/Cas9 carriers C1,C2,C3 according to exon 2 of Mongolian cattle MSTN gene.The C1 was successfully constructed and had activity by sequenceing and identifing in vivo,which can be employed to the subsequent cell transfection experiment.In this research,C1 was transfected into bovine fetal fibroblast cells by electroporation.Then we foster single cell by successive dilutions and pipette method.The positive cells were selected after cell proliferation.Our results showed that the 37 single cell strain lacked the single base in targeted areas,which could not encod right proteins.It can be used as donor cells for somatic nuclear transfer.The research provides an experimental material for subsequent production of marker-free transgenic cattle.Moreover,these results provide a basis for the production of superior traits,high meat production rate Mongolian cattle.