Establishment Of Knock-in Transgenic C2C12 Cell Line That Can Express Cas9 Protein In Muscle Specially

CRISPIR/CAS9 system is an important technology of gene editing.Studies have confirmed that the Rosa26 gene encodes a non essential RNA,which can be used as a foreign gene insertion site.In order to insert the sequence of myoblast specific expression of Cas9 protein into C2C12 cells,the CRISPIRJCas9 system was used to insert the Cas9 protein with MCK promoter and the fluorescent reporter gene through homologous recombination at the Rosa26 point of the mouse,and the positive cells were screened by purinomycin screening.The following results are obtained through this study.1.Construction of Rosa26-sgRNA vector.Oligo primers(gRNA)were designed according to the sequence of XbaI in mice Rosa26.The double stranded DNA sequence obtained after annealing was connected with the PX459 vector of BbsI mononuclease cut,and the vector Rosa26-PX459 was obtained.And Rosa26-PX459 was transfected into mouse fibroblastsused by Lipo2000,genomic DNA was extracted after 48 hours.Primers were designed to be used to perform PCR on the target site.The result of restriction enzyme digestion by Xbal showed three bands(not completely cut)in the PCR products transfected with the Rosa26-PX459 vector compared to the two bands in the wild type.The above results indicate that gRNA can guide Cas9 protein to gene editing on Rosa26 site.2.Construction of MCK-Cas9 vector.A 1358 bp fragment of mouse muscle-type creatine kinase(MCK)promoter containing SP1 and RBS regulatory elements was amplified by PCR and ligated with Rosa26-PX459 which excised the CMV promoter.Mouse muscle specific CRISPR/Cas9 vector Rosa26-PX459-MCK was successfully constructed.48 hours later DNA and RNA were extracted after the recombinant plasmid was transfected into C2C12 cells using Lipo2000.Three bands(not completely cut)appeared in the PCR product digested with Xbal.After being verified by T7E1 digestion,it was found that the PCR product could be cut into 2 small fragments.The agarose gel electrophoresis results were analyzed by gray scale and the mutation rate was 15%,which was lower than 24%of the CMV promoter.qPCR results showed that the Cas9 expression level of C2C12 cells transfected with Rosa26-PX459-MCK vector was 20%of that of C2C12 cells transfected with Rosa26-PX459 vector.3.Construction of mouse Rosa26 locus homologous arm targeting vector.In order to facilitate the observation and detection of subsequent experiments,To facilitate the observation and detection of subsequent experiments,a T2A sequence was added before the DsRed sequence,which was ligated downstream of the Rosa26-PX459-MCK vector Cas9 sequence.The MCK-Cas9-T2A-DsRed sequence was amplified by PCR from the recombinant plasmid and ligated to a Donor vector carrying the homologous arm around the mouse Rosa26 site to obtain the Donor-MCK-Cas9-T2A-DsRed vector.Using the same method,the CMV-Cas9-T2A-DsRed fragment of the original CMV promoter was ligated to the Donor vector to obtain the Donor-CMV-Cas9-T2A-DsRed vector.4.Screening of puromycin resistant cells and verification of gene insertion.When the homologous targeting vector was transfected into C2C12 cells or 293T cells,it was found that 293T cells and C2Cl2 cells transfected with the Donor-CMV-Cas9-T2A-DsRed vector could observe both red and green fluorescence,indicating that the vector enters the cell and Cas9 also could be expressed.293T cells transfected with Donor-MCKCas9-T2A-DsRed vector could only observe green fluorescence,while C2C12 cells can observe both red and green fluorescence.MCK promoter can initiate gene expression in C2C12.It did not work in 293T cells,indicating that the MCK promoter has muscle-specific expression activity.After Rosa26-PX459 vector co-transfected C2C12 cells with homologous targeting vectors 48h,green signal and red signal were observed respectively.After adding the lug/mL puromycin for 7 days,the genome was extracted.Near the insertion site,upstream and downstream primers were designed for the genome and the vector.The PCR results showed that both homologous targeting vectors could be integrated into the mouse genome Rosa26 loci.The above results indicated that the mouse Rosa26 gene site could be targeted through the CRISPIR/CAS9 system,and MCK-Cas9-T2A-DsRed and or CMV-Cas9-T2A-DsRed sequences could successfully be inserted into mouse C2C12 genome.the The method of exogenous gene knocking in mouse C2C12 cells was successfully established,which laid a foundation for the preparation of transgenic mice.