Method Establishment And Functional Verification Of Human SH2B3 Gene Knockout In Human Cells

Red blood cells,the main and most abundant cells in the blood,play an important role In the processes of oxygen transport and immunization.At present,the clinical transfusions mainly come from the society unpaid blood donation.However,it is unstable,and has some safety problems.There are some theoretical and experimental basis in the field of red blood cells induction by human embryonic stem cells and induced pluripotent stem cells,but the low induced efficiency makes the preparation cost high.In addition,the ethical and safety issues also limit the further use of stem cells in the field of red blood cells induction.Human umbilical cord mesenchymal stem cells,as a kind of adult stem cells,without ethical and safety problems,which makes it to be the ideal initial cells.Studies shown,knockout or suppressing the expression of SH2B3 gene can improve the efficiency of red blood cells induction in stem cells.In this study,we edited the gene of target cells by CRISPR/Cas9 technology.For the characteristics of the original cells,we knockout of the SH2B3 Gene of human umbilical cord mesenchymal stem cells by the strategy of Inserting two different fluorescent genes and two different resistant genes via CRISPR/Cas9-mediated homology-dependent DNA repair.For the purpose of establishing an efficient method for the induction of mature red blood cells by human adult stem cells.The main results obtained in this study are as follows:1.The knockout vector and homologous recombination filter carrier were constructedTaking the vector pX330 as the framework vector,the vector pSH2B3-KO which co-express the Cas9 protein with the nuclear localization signal and the sgRNA targetting the fifth exon of SH2B3 gene was constructed.A variety of molecular cloning techniques were used to complete the splicing of multiple components of the homologous recombinant screening vector pTGP,then further complete the construction of pTMN 、pTMP、pTEGP、pTEMN and pTEMP.2.The verification of the SH2B3 gene knockout system via CRISPR/Cas9-mediatedhomology-dependent DNA repair.In the 293 cells,it is preliminarily proved that the screening vector constructed above can work normally,and the corresponding fluorescence signal can be observed under the fluorescence microscope.Then,after the drug treatment for 4 weeds in the 293 cells which are co-transfected with vertors pSH2B3-KO and pTGP,both at the cellular level and molecular level,demonstrating that the strategy of knockout of the SH2B3 Gene by inserting two different fluorescent genes and two different resistant genes via CRISPR/Cas9-mediated homology-dependent DNA repair,is feasible.In this way,the target cell types can be selected through fluorescence signal or drug treatment,so as to avoid the tedious and time-consuming experiment operation of limited dilution monoclonal method.3.knockout of SH2B3 gene by CRISPR/Cas9Treating the HeLa cells with the above gene-knockout system vis CRISPR/Cas9-mediated homology-dependent DNA repair,after cultured in medium with puromycin and neomycin for two weeds,gain 19 clones via limiting dilution.The genome DNA and total RNA were extracted for PCR and RT-PCR assay.The PCR results show,all the clones can be detected the knock-in of reporter genes.Among the 19 clones,89.5% clones was detected with double knockout,57.9% clones was detected with double knockout via knock-in of reporter genes,42.1% clones was detected with double knockout via knock-in of reporter genes.The results of sequencing and RT-PCR show that the screening components have been accurately integrated into the genome,and can be further detected at the transcriptional level.Howener,when preliminary applying the SH2B3 knockout strategy vis homology-dependent DNA repair to the primary cells,human umbilical cord mesenchymal stem cells,the efficiency of electric transfection can reach about 30% by the method of electric transfection.Due to the insufficient proliferation ability of the transfected cells after drug screening,SH2B3 gene knockout cell lines have not been screened in the primary human umbilical cord mesenchymal stem cells yet.The drug screening concentration and restore conditions of human umbilical cord mesenchymal stem cells need to be optimized.