Generation Of DGAT1 Overexpressed Mice Via Microinjection

DGAT(acyl-CoA: diacylgly-cerol acyltransferase;)catalyzing the acylation of sn-1,2-diacylgly-cerol at the sn-3 position using an acyl-Co A substrate,and is the key enzyme to control the synthesis rate of triglycerides in adipocytes.The triacylglycerol is the major component of intramuscular fat.Increasing of triacylglycerol will contribute to the deposition of intermuscular fat.Therefore,DGAT1 can be used as candidate gene to improving the meat quality of livestock.CRISPR/ Cas9 technology is efficient gene engineering tool in farm animals.To produce the DGAT1 overexpressed transgenic mice,DGAT1 gene targeting vectors at Rosa26 locus by CRISPR / Cas9 s system were constructed,then injected into zygotes of mice.The results as follows:1.Construction of vector.The sgRNA/Cas9 targeting vector and DGAT1 vector were constructed.And they would be used in homologous recombinant of DGAT1 gene at mice Rosa 26 locus.Two vectors were verified correctly by enzyme digestion and sequence analysis.2.Identification of the vectorThe homologous recombinant vector of DGAT1 and targeting vector were transfected into C2C12 cells by electroporation transfection.The homologous recombination of exogenous genes was identifed by PCR.The results showed that the DGAT1 gene was knocked in mice Rosa 26 locus.3.Preparation and Identification of transgenic miceThe sgRNA / Cas9 plasmid and DGAT1 integrated vector were injected into mice's zygote by microinjection.314 embryos were obtained from injected 657 zygotes.33 mice were born after transplantation and identified by PCR.Two of them were positive mice,the positive rate was 6%.4.Detection of DGAT1 Gene expression in transgenic miceLevel of DGAT1 expression in transgenic mice muscles was detected by real-time quantitative PCR.It was found that the level of relative expression of mRNA in mice No.2 was 2.54 times higher than that in control mice,and in mice No.4 was 3.57 times higher than that in control mice.The level of DGAT1 protein expression was detected by Western Blot,the results showed that the level of relative expression of protein in No.2 mice was 1.25 times higher than that in control mice,and in the No.4 mice was 1.43 times higher than that in control mice.5.Determination of fatty acids in muscles of transgenic mice.The content of triglycerides and fatty acids in muscles of the transgenic mice were determined.The results showed that the content of both in transgenic mice muscle is increase compared with a control group.It was proved that over expression of DGAT1 gene increased the accumulation of intramuscular fat in mice.The animal model of DGAT1 overexpression was sucessfully establishmented.Therefore,this study has laid a foundation for the domestic animal breeding.