Construction And Invesigation Of Zfp217 Gene Knockout Cell Lines And Mouse Models Using CRISPR/Cas9

Studies have indicated that numerous transcription factors contributed to regulating the process of adipogenesis.Over the past two to three decades,although most of the transcription factors have been identified to participate in the regulation of adipose Tissue development,it is not yet fully understood.but the mechanisms remain to be further explored.A new transcription factor Zfp217 that may involved in the regulation of adipogenesis,was identified by RNA sequencing and bioinformatic analysis during SV cells adipogenic differentiation at the early stage.Thus,we posted a hypothesis predicting that the effect of Zfp217 on adipogenic differentiation of 3T3-L1 preadipocyte.In this study,to test this hypothesis,we constructed Zfp217 gene knockout monoclonal cell lines by using CRISPR/Cas9 gene editing technique with 3T3-L1 preadipocyte as the research material.To further verify the relationship between Zfp217 and fat formation,we established Zfp217 gene knockout mouse by CRISPR/Cas9 gene editing technique.Currently,the main research results are as below:?1?Screening of sgRNA with cleavage activity.First of all,four sgRNAs for first exon of Zfp217 gene were designed by sgRNAcas9₃.0.5 software and http://crispr.mit.edu/website,and we successfully constructed CRISPR/Cas9 system?pX458?vector.Then,we preliminary identified sgRNA-2?sgRNA-3 and sgRNA-4 have cleavage activity by T7E1 digestion and DNA sequencing.?2?To investigate the effect of Zfp217 on adipogenic differentiation of 3T3-L1 cells,we constructed Zfp217 gene knockout monoclonal cell lines.The pX458 vector containing sgRNA-4 was transfected into 3T3-L1 cells,so homozygous monoclonal cell lines that knockout Zfp217 were obtained.We observed Zfp217 gene knockout monoclonal cell lines that be stained with Oil Red O,showed lipid droplets in the homozygous Zfp217 knockout cell lines were significantly lower than that in the control group;Western Blot analysis of adipogenic marker gene expression revealed that knockout Zfp217 significantly inhibited the expression of PPARy and aP2 protein.These above results indicate that knockout of Zfp217 inhibited adipogenic differentiation of 3T3-L1 cells.?3?Establishing Zfp217 gene knockout mouse.First,PCR amplification of sgRNA-2 and sgRNA-4 in vitro transcription template that by the in vitro transcription kit and RNA purification kit.sgRNA-2,sgRNA-4 and Cas9 mRNA were microinjected into the fertilized eggs,then the injected fertilized eggs were cultured and developed into blastula.The embryos were transplanted into the uterus of the fake pregnant mothers and 15 mouse were obtained 21 days later.?4?Identifying Zfp217 gene knockout positive mouse.The results of PCR genotyping and DNA sequencing confirmed that we obtained 10 positive mouse in F0 generation,and successfully established Zfp217 gene knockout mouse.