Objective:To investigate the expression of target genes and myoblast factors in C2C12 mouse myoblasts after XLOC_015548 gene knockout or overexpression,and to construct XLOC_015548 gene editing muscle cell model so as to provide a basis for future experiments.Methods:According to RNA-seq results,the XLOC_0 15548 knockdown and overexpression lentivirus vectors were constructed,and the C2C12 myoblast cell line was transfected to prepare the myoblast model of XLOC_015548 gene editing.Fluorescence microscope was used to observe the expression of green fluorescent protein(GFP)in cells infected with lentivirus.When XLOC_015548 was knocked out or overexpressed,the expression levels of XLOC_015548 and myogenic genes were detected by RT-qPCR and Western Blot.Results:Myoblasts were transfected with gradient multiplicity of infection(MOI)of 1,2,5,10,20 and 30.After screening by purinomycin,XLOC_015548 gene editing myoblasts with MOI being 20 were successfully constructed.RT-qPCR showed that when XLOC_015548 was overexpressed,the expression of Myod increased,and when XLOC_015548 was knocked down,the opposite result was obtained.Western Blot indicated that overexpression of XLOC_0 15548 could improve the expression levels of Myod and Myogenin,while knockdown results showed opposite results.Conclusions:A novel long chain non-coding regulator XLOC_015548 gene editing myoblast model is constructed.The expression level of XLOC_0 15548 is preliminarily verified in each cell model,and its correlation with myogenic differentiation factor is invetstigated.The cell model can be used for further basic research on orthopaedic diseases such as denervation muscular atrophy.
关键词
长链非编码RNA/成肌细胞分化/慢病毒/RNA测序
Key words
Long Non-coding RNA/Myoblast Differentiation/Lentivirus/RNA-seq
万方数据