慢病毒介导长链非编码RNA XLOC_015548基因编辑的C2C12细胞模型建立

Lentivirus-mediated C2C12 cell model for gene editing of long non-coding RNA XLOC_015548

目的:探讨长链非编码RNA XLOC_015548基因敲减或过表达后C2C12小鼠成肌细胞中目的基因及成肌分化因子的表达情况。构建XLOC_015548基因编辑成肌细胞模型,为今后进一步实验提供基础。方法:根据RNA测序结果得出相关序列,拟构建XLOC_015548敲低及过表达慢病毒载体,并对C2C12成肌细胞系进行转染,制备出XLOC_015548基因编辑成肌细胞模型。使用荧光显微镜观察慢病毒感染细胞后绿色荧光蛋白质的表达。当XLOC_015548被敲除或过表达后,采用RT-qPCR及Western Blot检测XLOC_015548及成肌相关基因的表达量。结果:采用1、2、5、10、20、30的梯度感染复数(MOI)对成肌细胞进行转染,经嘌呤霉素筛选后,成功构建以MOI为20感染条件的XLOC_015548基因编辑成肌细胞模型。利用RT-qPCR初步检测出过表达XLOC_015548时,肌源性分化因子Myod的表达量升高,敲低XLOC_015548时则得到相反的结果。Western Blot实验提示过表达XLOC_015548可以提高Myod、Myogenin蛋白表达量,敲低后则得到相反的结果。结论:本研究构建了一种新的长链非编码调节因子XLOC_015548的基因编辑成肌细胞模型,初步验证了各组细胞模型中XLOC_015548的表达量,并探究其表达量与肌源性分化因子的相关性,该细胞模型可用于后续对失神经肌萎缩等骨科相关疾病的基础研究。

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_015548 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_015548 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_015548 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.