Establish The Differentially Methylated Region CGI-2 Knockout Mice By Using CRISPR/Cas9 Gene Editing Technique

The Dlk1-Dio3 imprinting region is an important imprinting gene cluster in the mouse genome.There are multiple protein-coding genes,long non-coding RNA genes,and numerous micro RNA and sno RNA genes.The imprinted expression of this imprinted region gene is important for embryonic development.In-depth study on the expression regulation and mechanism of the gene in this imprinting region is of great significance for revealing the role of imprinted genes in embryonic development and obtaining high-quality i PS cells.The current study is limited to the regulation of gene expression in the imprinted region by three known paternally methylated differentially methylated regions(IG-DMR,Gtl2-DMR,and Dlk1-DMR).Therefore,the search for and identification of new differentially methylated regions is of great significance for further analysis of the gene regulation mechanism and molecular mechanism of this imprinted region.Combined with a new differential methylation region CGI-2 found in the previous study of this laboratory,this project used a new generation of gene editing technology CRISPR/Cas9 to establish differential methylation region CGI-2 knockout mice.We first designed and prepared templates for in vitro transcription of sg RNAs and Cas9 in vitro transcription templates,which were then microinjected into mouse fertilized eggs and cultured into blastocysts and transplanted into the uterus of pseudopregnant mother mice.,successfully obtained a Founder(first mouse).Finally,64 blastocysts were transplanted and 5 newborn mice were obtained.After identification,there were 1 CGI-2 single allele knockout mice with a total positive rate of 20%.We bred the Founder mice with wild-type ICR mice to produce CGI-2 single allele knockout mice in the offspring and then mated with Founder mice to obtain CGI-2biallelic knockout mice.We compared the changes in signs and body weights of three genotype mice(wild-type mice,CGI-2 binomimal knockout mice,and CGI-2 single allele knockout mice)and found that they were born one day later.At the time,there was no significant difference between the three genotypes of the mice,and from the first week to the fourth week after birth,the body weight and size gradually showeddifferences.The performance was as follows: the weight and body of the wild type mice were larger,followed by CGI-2 biallelic knockout mice,and finally CGI-2 single allele knockout mice.The results of tissue HE staining of three genotype mice showed that compared with wild-type mice,CGI-2 ambiguous knockout mice and CGI-2 single allele knockout mice showed a large number of inflammatory cells in the alveolar stroma.There was haemorrhage in the alveolar space and lung interstitium,which belonged to bronchus associated with interstitial pneumonia and lung consolidation;slight changes in the morphology of hepatocytes;disappearance of glomerular structure and infiltration of surrounding inflammatory cells;and necrosis of necrosis in the renal interstitium.The establishment of the model provides an important tool for the follow-up study of the function and mechanism of CGI-2,and can also provide a technical reference for the study of other important genes in vivo.