Constructing RBM24 Knockout Mouse Embryonic Stem Cell Lines By CRISPR-Cas9 Technology

In this study,a technical platform based on RBM24 gene knockout was proposed to complete the establishment of RBM24 high-efficiency specific knockout method and the exploration of related cardiac development content.At the same time,the related bioinformatics analysis is combined to explore the contents of various aspects.This topic is to set up efficient and specific CRISPR(Clustered regularly interspaced short palindromic repeats)technology platform,specifically,the platform can knockout the exon 1 of the gene be called RBM24 in human and mouse genomes,and the method of using PCR and flow cytometry screening and purification RBM24 specificity knockout cell lines.PCR and flow cytometry were used to screen and purify RBM24 specific knockout cell lines.The CRIPSR-Cas9 system is mainly composed of two parts:the first part is the recognition effect of single-guide RNA,the other part is the Cas9 protein that is used to cutting.Based on the hek-293t cell line,we designed and verified the feasibility of the Cas9 system guided by dual-gRNA and its related identification methods.CRIPSR-Cas9 knockout system guided by the dual-gRNA already has the characteristics of highly shear efficiency specificity and easy to operate,also facilitate follow-up tests,simply by PCR method can easily detect RBM24 knockout situation,Specific gRNA is designed for each site in this project.This study targets RBM24 gene,using the CRISPR/Cas9 technology and the method of mouse embryonic stem cells to myocardial cell differentiation,to establish a platform to research on the molecular mechanisms of RBM24 deletion embryonic stem cells and myocardial cell development.Firstly,the gRNA expression vector of dual-target RBM24 gene was designed and constructed,the highly purified and non-endotoxin Cas9 and gRNA vectors were established,the technical method for the introduction of cells were set up.Furthermore,we expect to optimize the detection scheme.The objectives include the following points.(a)To construct a dual-gRNA guided CRIPSR-Cas9 vector,and to evaluate its feasibility and subsequent detection methods.(b)Using the CRIPSR-Cas9 gene editing technique,RBM24 gene knockout mouse embryonic stem cell(mESC)stable cell line was constructed.(c)To explore whether the cell pluripotency of the mESC system with the RBM24 gene knockout was affected after electroporation and flow cytometry.(d)To explore whether the ability of the embryoid(EB)formed by mESC to differentiate into myocardial cells will change after RBM24 gene knockout.In this paper,molecular cloning technology is used to construct pX458M(pX459M)vector for knockout RBM24 gene exon 1.Then the pX458M(pX459M)-RBM24KO vector with dual-gRNA is transferred to mESC.The next step was to use flow cytometry to select the cell lines with green fluorescent protein(GFP)positive.PCR was used to determine whether the cell lines were successful,and the results of cell lines were identified by sequencing analysis.Morphological observation,karyotype analysis and fluorescence staining were performed on successfully constructed cell lines,and the biological multipotency of mESC after RBM24 knockout was detected.In vitro culture,the mESC-RBM24KO cell line cultivates the embryoid body(EB),and then we induce EB to differentiate into myoblasts,the total protein of cells in the 0,6,9,12 days after EB differentiation was collected,and the RBM24 knockout was detected by the western blotting.The results of plasmid sequencing analysis showed that the pX458M(pX459M)-RBM24KO vector with dual-gRNA was successfully constructed for the hek-293t cell lines form human species and mES cell lines.In mESC of RBM24 gene knockout,cells showed the typical morphology of mouse embryonic stem cells.In addition,the cell maintains normal karyotype,and the cell pluripotent marker OCT4 also presents high expression.Western blotting detection and PCR detection results show that the vector has a remarkable effect,RBM24 has been completely eliminated in RNA and protein levels.The specific gene editing of mouse embryonic stem cells can be achieved by using CRIPSR-Cas9 gene editing technology.mESC,which was knocked out of the RBM24 gene,still maintains the pluripotency of stem cells,but has lost the ability to differentiate into cardiomyocytes.We get mESC which RBM24 genes knocked out can be used to further explore the molecular mechanism of myocardial cell development,it provides a new research system for the treatment of congenital cardiomyopathy and other related diseases caused by RBM24 gene abnormalities.