The Generation Of Gtl2 Transcriptional Terminated Knock-in Mouse Model With Easi-CRISPR Technology

Long non-coding RNA(lncRNA)Gtl2 is an imprinted gene located on the Dlk1-Dio3 imprinted gene cluster.It has the imprinted pattern of maternal expression and paternal imprint,which plays an important role in the development of mouse embryos.At present,there is no clear conclusion on the regulatory role of lncRNA Gtl2 in mouse embryo development,and two gene knockout studies on Gtl2 gene have given conflicting results.Although the embryonic lethal phenotype appeared in both articles,proving that Gtl2 is an important gene for mouse embryonic development,the reported lethal time and lethal mechanism are different from each other,and largescale gene knockout will delete Gtl2-DMR,mi RNA and other important elements.In addition,a large number of studies have shown that the gene sequence of lncRNA has a regulatory effect,and gene knockout will confuse the function of the gene sequence and lncRNA itself,bringing unreliable results and not suitable for the study of lncRNA function.In this study,a poly A transcription termination method combined with Easi-CRISPR technology was used to construct a mouse model of Gtl2 transcription termination,which achieved the same effect as gene knockout without deleting any DNA sequence.In-depth analysis of Gtl2 gene sequence and transcript information,fix the selection range of sg RNA in the first three exons and first two intron sequences,and select the lower active non-functional DNA region as the sg RNA targeting site,avoiding damage to control elements.Assemble the ss DNA donor according to the donor design principles required by the Easi-CRISPR technology,verify the ability of sg RNA to guide the Cas9 protein editing genome through in vivo and in vitro editing,and measure the transcription termination ability of the 3×poly A sequence in the ss DNA donor.Using pronuclear injection to edit the fertilized eggs of B6D2F1 strain mice,surgically transplanted blastocysts into the uterus of surrogate mice,successfully obtained 5 F0 generation mice with positive inserts,no off-target detected,successful GTR-poly A mouse model was constructed.Genetic hybridization showed that no homologous mice were born,and the proportion of genotypes of the born pups seriously deviated from Mendelian laws of inheritance.In addition,this study optimized the gene insertion system and explored the effects of microinjection methods and injection timing on knock-in efficiency.Five injection methods that may improve the efficiency of gene insertion were designed: fertilized egg cytoplasmic injection(1CC),fertilized egg pronuclear injection(1CN),2-cell phase dual cytoplasmic injection(2CDC),2-cell phase mononuclear injection(2CMN).And,2-cell stage binuclear injection(2CDN).It is concluded through three repeated experiments in each group that the pronuclear injection method can achieve a gene insertion efficiency of up to 67%,and the effect of 2-cell microinjection on fertilized eggs will be better than that of fertilized eggs cytoplasmic injection.In summary,this subject produced a GTR-poly A mouse model.The results of genetic hybridization experiments seriously deviated from the theoretical ratio,suggesting the important role of lncRNA Gtl2 and laying the foundation for confirming the function of lncRNA Gtl2 on mouse development.