The Research Of CGI-2 Regulate Gene Expression In Dlk1-Dio3 Imprinted Domain

The Dlk1-Dio3 imprinted domain is located on mouse chromosome 12qF1 and human chromosome 14q32. The Dlk1-Dio3 imprinted domain has an important function to regulation growth and development of emrtyo and placenta, and it also effects on body’s metabolism and the functions of brain. Those genes in Dlk1-Dio3 imprinted domain are dynamic in process of growth and development. CGI-2 is around the miR-341 and miR-1188. CGI-2 is the first maternally methylated DMR was found in the mouse Dlk1-Dio3 imprinted domain, and it displays enhancer-blocking activity dependent upon the CTCF binding site. Thus, this paper aims to study the change of genes expression in the Dlk1-Dio3 imprinted domain when the CGI-2 is knocked out by the CRISPR/Cas9-mediated genome editing technology.Firstly, our research aims to choose the appropriate cell clone to be used in the knockout experiment by analyse the expression level of genes in the Dlk1-Dio3 imprinted domain. Through the analysis, we choose three cell clone, they are the N2 a, which has a high expression level of genes in the Dlk1-Dio3 imprinted domain, the Hepa1-6, which has a low expression level of genes in the Dlk1-Dio3 imprinted domain, and the NIH3T3, which is a normal cell clone deffer form the cancer cell. In addition, we design two sets guide RNA used to CRISPR/Cas9-mediated genome editing by the guide RNA design site. And the recombinant vectors are structured by connecting, inversion and selecting.Secondly, the CGI-2 knockout experiment is in progress in N2 a, and the knockout result is confirmed by sequence alignment. The befitting guide RNAs are choosed by anaylse the knock out efficiency and the length of knockout fragment. the CGI-2 is knocked out by CRISPR/Cas9-mediated genome editing in the N2 a, Hepa1-6 and NIH3T3 respectively, then extracting RNA after knockout result confirmation. Genes expression level variation tendency are judged by real-time quantitative analysis. As the result, the expression level of Dio3 has no obvious change, the expression level of Dlk1 has no significant downward trend, the Mirg expression level of has different variation tendency in different cells, and the expression level of Gtl2, Rtl1, Rian, Meg8, Irm, AB063319 is rise significantly. In conclusion, deletion of CGI-2 lead to the genes expression level change in the Dlk1-Dio3 imprinted domain, it means that CGI-2 has the function of regulation the genes expression in the Dlk1-Dio3 imprinted domain.Finally, the methylation status of the differentially methylated region(DMR) Dlk1-DMR, Gtl2-DMR, IG-DMR and CGI-1-DMR in the Dlk1-Dio3 imprinted domain is analyzed between the CGI-2 knockout sample and the CGI-2 not knockout sample by bisulfite sequencing test. The result shows that Dlk1-DMR, Gtl2-DMR, IG-DMR and CGI-1-DMR are highly methylated,and the methylation status of four differentially methylated regions have no change.To sum up, in the Dlk1-Dio3 imprinted domain, the lack of CGI-2 will lead to change the genes expression level, and the variation tendencies of genes in different cells are coincident when CGI-2 knockout. The CGI-2’s regulate genes expression in the Dlk1-Dio3 imprinted domain, it has nothing to do with other differentially methylated regions.