Preliminary Study On GLUT2 Gene Editing System Mediated By CRISPR/Cas9

GLUT2 is the major sugar transporters in chicken intestinal. It's expression directly affects the absorption of carbohydrates. To achieve its biological functions, it may dependent on multiple transcription factors, such as the transcription factors of USF1, GATA2. However, the transcription factor binding sites are unclear. To determine the transcription factor binding sites, we choose a new techniques CRISPR/ Cas9. It is a kind of new gene editing technology, which developed from the immune mechanism of archaea resisting the invasion of exogenous nucleic acid. It is dependent on RNA, which can be fixed to edit the specific gene sequence. Since 2013, it has been successfully applied to a variety of plants, animals and cells. At present, the application of CRISPR/Cas9 technology in chicken is rarely reported.. The purpose of this test is mainly to build the Cas9 editing platform in chicken cells and explore its application in chicken.Build the Cas9 editing platform in chicken cells is mainly composed of three parts, culture chicken cells, construct the targeting vector and transfection. The experiment was repeated by a large number to explore the three steps. Firstly, established the basic conditions of culturing CEF. Including isolation and culture,subculture, cryopreservation, recovery, determined the growth status and compared the different methods.Then explore the conditions of targeting vector, regard the px458 vector as a basic skeleton, and find the two potential binding sites of transcription factors to construct the px458-GLUT2 vector. And transfected it into 293T cells.Test results showed that:different preparation methods were successfully in cultured chicken embryo fibroblasts, however the CEF by trypsin digestion were grown faster and the period was shorter; And the growth rate of CEF which from chicken embryo by artificial insemination was faster and stronger than the natural insemination chicken embryo. Verified by PCR and sequencing, we found that px458-GLUT2 vector was successfully constructed. And successfully transfected it into 293T cells. It was initially set up a Cas9 editing platform in the cell.