Production Of Site-directed Integration Of FABP4 And MSTN Point Mutation Transgenic Mouse And Beef Cattle

Mystatin(MSTN)acts as a negative regulator of skeletal muscle development and growth,and its expression can significantly inhibit muscle growth.The MSTN is highly conserved during mammalian evolution,and its loss of function causes muscle overgrowth in the animals,resulting in a double muscle(DBM)phenotype.Naturally mutated MSTN have been discovered in numerous species including cattle,sheep,goat,horse,pig,dog,rabbit.Mutations in the MSTN gene have also been achieved in many species by gene editing techniques.In modern livestock breeding,the mutation of MSTN mediated by nuclease technology can produce a double muscle phenotype with high carcass yield.However,animals with a DBM phenotype have a relatively reduced fat content due to abnormal increased muscle in the body,which directly affects the quality of the meat.As a lipid protein chaperone,adipose fatty acid-binding protein(FABP4)has high affinity for long-chain fatty acids and promotes its dissolution and transport in cells.FABP4 is involved in fat deposition in animals,which is closely related to subcutaneous fat content and intermuscular fat(IMF)content.Compared with foreign high-quality of beef cattle breeds,our native have lower meat yields and relatively poorer quality.To solve the problem,the FABP4 related to IMF deposition was site-directed integrated into the MSTN locus of Luxi cattle by gene editing technology,and the G938 A point mutation was introduced simultaneously.The FABP4 was specific expressed in skeletal muscle tissue by means of an endogenous promoter,which increases the carcass yield and IMF content.The main contents and results of this research are as follows:1.A sgRNA targeting site mediated by CRISPR/Cas9 system was screened near the stop codon site of the mouse MSTN gene,and the corresponding homologous recombination targeting vector was constructed.F0 generation positive transgenic mouse were obtained by pronuclear microinjection technique.The transgenic mouse were expandedand and identified by Junction PCR,Long-range PCR and Southern blot.The identified heterozygous and homozygous transgenic mouse models of site-directed integration of FABP4 and MSTN point mutation can be used to subsequent phenotypic studies.2.Wild-type mouse,heterozygous and homozygous transgenic mouse with the same age were dissected,and the muscle morphology in major parts of the body were compared.The homozygous transgenic mouse had an obvious double muscle phenotype.Histological examination of the main organs and skeletal muscle tissues showed that the double muscle phenotype of the homozygous transgenic mice was caused by the increased cross-sectional area of the muscle fibers.The detection of FABP4 protein and triglyceride content in skeletal muscle tissues showed that the FABP4 protein content increased significantly and promoted the deposition of triglyceride in muscles.3.A region capable of fusion expression of exogenously inserted genes was found in the MSTN locus of Luxi cattle.The potential off-target statistics and analysis of all sgRNA targets in this region were performed by CHOPCHOP and Cas-OFFinder online analysis software.Four candidate sgRNA targets with relatively few off-target sites were obtained.The corresponding CRISPR/Cas9 cleavage vector was constructed,and the cleavage activity detection and off-target effect detection were performed in the Luxi cattle fetal fibroblasts,and finally the sgRNA1 target was determined to have relatively high cleavage efficiency with no off-target effect occurred.4.For the sgRNA1 target,a site-directed integration of FABP4 and MSTN point mutation homologous recombination targeting vector was constructed,and the corresponding CRISPR/Cas9 cleavage vector were co-transfected into Luxi cattle fetal fibroblasts.The cell colonies were screened with puromycin,and six biallelic targeted positive colonies were identified by junction PCR,long-range PCR and Southern blot.5.Using the above positive cell colonies as nuclear transfer donor cells,the transgenic cloned embryos were obtained by somatic cell nuclear transfer technology.The randomly selected transgenic cloned embryos were subjected to PCR detection,eliminating the possibility of mixing non-gene targeting cells in the donor cells.The well-developed cloned blastocysts were transfer to recipient cattle uterus,and a total of 19 recipient cattle to maintain pregnancy up to now.In summary,this research established the site-directed integration of FABP4 and MSTN point mutation transgenic mouse model to verify that the homozygous point mutation of MSTN can cause the animal to produce double muscle phenotype,and FABP4 specific expression can increase skeletal muscle tissue triglyceride content.In the meanwhile,the CRISPR/Cas9 system was used to achieve the site-directed integration FABP4 and MSTN point mutation transgenic beef cattle,which provided important breeding materials for the cultivation of high-yield and high-quality beef cattle breeds in our country.