CRISPR/CAS9 Mediated DGAT1 Gene Knock-In At MSTN Locus In Mouse

MSTN (Myostatin) is the negative regulator in skeletal muscle growth, the MSTN gene knock-out could enhance muscle mass in animal models. DGAT1 is a glycerol acyltransferase, the enzyme is closely related to fat metabolism and lipid deposition in tissues. CRISPR/Cas9 recognise the DNA specific sites by RNA, the core RNA-guided Cas9 endonuclease in the DNA has been harnessed to realize gene mutation, DNA deletion and insertion. The objective of this study is to get MSTN knock-out and DGAT1 gene knock-in in mouse embryonic fibroblast cells by CRISPR/Cas9 technology.The results will provide experimental bases for the production of animals.The results are as follow:1 The gRNA in CRISPR/Cas9 system and knock-in vector CAG-DGAT1 were successfully constructedgRNA vector was successfully constructed for MSTN in mouse, The results of surveyor mutation detection proved that Cas9 can efficiently cut DNA at specific sites under the guidance of the gRNA. DGAT1gene knock-in vector was proved correct by PCR, enzyme digestion and sequencing.1. The improvement of mouse fibroblast cells and mouse embryonic stem cells transfected selectionElectroportion and lipofectin transfection were compared in two different cells. The results showed that electroportion was significantly higher than lipofectin transfection in mouse fibroblasts cells, lipofectin transfection was significantly better than electroporation in mouse embryonic stem cells.2. Detection of genetically modified cells of DGAT1DGAT1 gene was confirmed that it has been integrated into the MSTN site by the methed of PCR.The expression of target gene and associated gene were detected by the methed of real-time PCR. The results showed that the expression level of DGAT1 in the positive cells was 32.76 times higher than the control group. With the increase of the content of DGAT1, the expression of genes which related to the metabolism of fat was increased in different degrees, acetyl coenzyme A was increased 1.58 times, fatty acid binding protein 4 was increased 7.11 times and DGAT2 was increased 2.93 times. It showed that increased DGAT1 will promot the synthesis of fat. The expression of uncoupling protein I was increased 2.17 times. Because the DGAT1 gene was inserted into MSTN site, the expression of muscle related genes was also detected in this study. The results showed that the expression of Foxo1 was increased 23.53 times,Mef2c was reduced 0.66 times,the expression was down regulated...