Construction of Lrtm1 gene knockout C2C12 cell line using the CRISPR/Cas9 system

Abstract:

Objective：To construct Leucine-rich repeats and transmembrane domains 1（Lrtm1） gene knockout C2C12 cell line using the CRISPR/Cas9 technique and to provide an experimental basis for studying the functions of the Lrtm1 gene. Methods：Three pairs of small guide RNA（sgRNA） targeting the Lrtm1 gene were designed and inserted into the vector pCRISPR-LvSG06，and the recom－binant plasmid pCRISPR-LvSG06 containing sgRNA was packaged by the lentiviral packaging system. Then the C2C12 cells were infected with the virus and screened by puromycin，and RNA was extracted from the screen-positive cells and reversely transcribed into cDNA. The Cas9 primer was designed using the cDNA as the template to verify the expression of Cas9 in C2C12 cells by PCR and to confirm that the C2C12 cells were successfully infected by the virus. Monoclonal cells were collected by the 96-well plate method，and the amplified monoclonal cells were used to extract genomic DNA and investigate the Lrtm1 gene-related sequences for comparison with the wild-type Lrtm1 gene，so as to identify the monoclonal cell line with successful gene knockout. After that，the stable Ltm1 gene knockout cell line was induced to differentiate into myoblasts and its expression of myosin，which was the marker of successful myogenic differentiation，was determined. Besides，the expression of transcription factor PAX7 mRNA and H3K27me3 protein was determined by RT-PCR and Western blot，respectively. Results：The sequencing results showed the successful insertion of the sgRNA into the plasmid vector and Lrtm1 gene knockout in clones A and C. The expression of both myosin protein as the myogenic differentiation marker and PAX7 mRNA as the myo－genic transcription factor decreased，while the expression levels of H3K27me3 protein in both the 72-hour and 96-hour differ－entiation groups were higher than those in the wild-type group. Conclusion：The CRISPR/Cas9 technique can be used for Lrtm1 gene knockout and successfully constructing the Lrtm1 gene knockout C2C12 cell line. Lrtm1 gene knockout can inhibit the myogenic dif－ferentiation of C2C12 cells and the expression of PAX7 mRNA，and the reason for the reduced expression of PAX7 mRNA may be at－tributed to the increased level of H3K27me3.