Generation And Identification Of EpCAM-regulated Glycolipid Metabolic Mouse Model Based On Cas9 Gene Modification Technology

Background:Glycolipid metabolic disease(GLMD)is a general term for a series of diseases caused by disorders of glucose metabolism and lipid metabolism.At present,researches on GLMD is mainly performed through the liver.The liver mainly functions as a “hub” in the entire life activities and can coordinate other organs functions.The liver and the intestine develop from the same germ layer at the development stage and play an important role in regulating metabolism,traditional Chinese medicine has many multiple levels and multi-targets in the treatment of glycolipid metabolic disorders.The epithelial cell adhesion molecule,EpCAM,has been shown to play an important role in maintaining the pluripotency of embryonic stem cells,and in many adult stem cells such as hepatic stem cells.EpCAM is highly expressed in mouse embryonic hepatic stem cells,low or no expressed in mature hepatocytes,abnormally expressed in hepatocytes of liver disease caused by hepatic damage such as hepatic fibrosis,liver cirrhosis,hepatic carcinoma.When EpCAM is missing,both human and mouse intestine will have the disease-congenital tufting enteropathy;In early studies,the tight junction structure of which the EpCAM-targed in the small intestine was changed,and this change would affect the intestinal function and thus regulate the liver,the earlier research has found that EpCAM can regulate glycolipid metabolism,but the specific mechanism is not yet clear.The CRISPR/Cas technology is a newly emerged genome editing tool that enables RNA-directed DNA recognition and editing.The CRISPR-Cas9 system has been successfully applied to plants,bacteria,yeast,fish and mammalian cells and is currently the most efficient genome editing system.The stable genetic EpCAM knockout mice were obtained by using the latest gene editing technology-CRISPR/Cas9,and the gene model mice were studied to explore the function of EpCAM intervention in glycolipid metabolic disease,and study its mechanism under the guidance of Chinese medicine theory.Objective:1.Using CRISPR/Cas9 technology in vitro to construct the carrier combined with electroporation,embryo transfer,mouse passaging and other technologies to obtain stable genetic EpCAM knockout mice.2.To validate the gene knock-out effect of EpCAM knockout mice.3.To study the effect after EpCAM gene deletion on the intestinal tract differentiative capacity,and then to explore the regulation mechanism of glycolipid metabolism.Method:1.Production of EpCAM knockout mice: Cas9 mRNA and gRNA vectors were constructed in vitro.F0 mice were obtained by electroporation and embryo transfer to identify the genotypes.Hybrid genotype mice and C57BL/6J mice were selected.The F1 mice were crossed and their specific sequences were identified.The male and female mice with completely identical knockout sequences were selected and then self-bred to obtain F2 mice.The F2 mice with stable genetic traits were selected for breeding and the homozygous knockout mice among them were selected for analysis.2.Gene knock-out validation of EpCAM knockout mice: Phenotypic analysis of EpCAM knockout mice,intestinal morphological and histochemical analysis,followed by Real-time PCR detection of intestinal tight junction molecules,and binding with immunohistochemical detection and analysis.3.Study on the differentiation ability of mouse intestinal cells after knock-out of EpCAM gene: Real-time PCR was used to detect the key marker of intestinal epithelial differentiation in EpCAM knockout mice and normal mice,and the changes of expression were analyzed by immunohistochemistry.Result:1.The CRISPR/Cas9 technique was used to successfully create an EpCAM knockout mouse model and determined the correct knockout through a series of gene identifications.At the same time,the heterozygous genotype mice were allowed to stably breed and inherit the genetic traits.2.Intestinal detection of EpCAM knockout mice revealed a significant change in phenotypes and disturbances in the expression of related linker molecules,and changes in intestinal cell differentiation and other related factors.Conclusion:The CRISPR/Cas9 gene editing technology was firstly used to create an EpCAM knockout mouse model to further study the function of EpCAM gene in mice.It was also found that EpCAM may regulate glucose and lipid metabolism by regulating intestinal cell differentiation.