Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA.
Stable Knockdown of Genes Encoding Extracellular Matrix Proteins in the C2C12 Myoblast Cell Line Using Small-Hairpin (sh)RNA.
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DOI：
10.3791/60824
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发表时间：
2020-02-12
期刊：
影响因子：
--
通讯作者：
Hubmacher D
中科院分区：
文献类型：
--
作者：
Taye N;Stanley S;Hubmacher D
细胞外基质(ECM)蛋白对骨骼肌发育和动态平衡至关重要。C2C12成肌细胞中ECM蛋白编码基因的稳定敲除可用于研究这些蛋白在骨骼肌发育中的作用。在这里，我们描述了一种耗尽ECM蛋白ADAMTSL2的协议作为例子，在C2C12细胞中使用小发夹状(Sh)RNA。转染shRNA载体后，用嘌呤霉素分批筛选稳定的细胞。我们进一步描述了这些细胞系的维持和通过mRNA表达、蛋白表达和C2C12分化进行的表型分析。该方法的优点是相对快速地产生稳定的C2C12基因敲除细胞，并在耗尽细胞培养液中的血清后可靠地将C2C12细胞分化为多核肌管。C2C12细胞的分化可以通过明场显微镜和通过测量典型的标志物基因的表达水平来监测，如MyoD、mygenin或myosin Heavy Chain(MyHC)，表明C2C12成肌细胞分化为肌管的过程。与带有小干扰(Si)RNA的基因的瞬时敲除相反，在C2C12分化或肌管成熟过程中后期表达的基因可以通过产生稳定表达shRNA的C2C12细胞来更有效地靶向。该方法的局限性在于基因敲除效率的可变性，这取决于可通过使用基于CRISPR/Cas9的基因敲除策略克服的特定shRNA，以及应考虑的shRNA的潜在脱靶效应。
Extracellular matrix (ECM) proteins are crucial for skeletal muscle development and homeostasis. The stable knockdown of genes coding for ECM proteins in C2C12 myoblasts can be applied to study the role of these proteins in skeletal muscle development. Here, we describe a protocol to deplete the ECM protein ADAMTSL2 as an example, using small-hairpin (sh) RNA in C2C12 cells. Following transfection of shRNA plasmids, stable cells were batch-selected using puromycin. We further describe the maintenance of these cell lines and the phenotypic analysis via mRNA expression, protein expression, and C2C12 differentiation. The advantages of the method are the relatively fast generation of stable C2C12 knockdown cells and the reliable differentiation of C2C12 cells into multinucleated myotubes upon depletion of serum in the cell culture medium. Differentiation of C2C12 cells can be monitored by bright field microscopy and by measuring the expression levels of canonical marker genes, such as MyoD, myogenin, or myosin heavy chain (MyHC) indicating the progression of C2C12 myoblast differentiation into myotubes. In contrast to the transient knockdown of genes with small-interfering (si) RNA, genes that are expressed later during C2C12 differentiation or during myotube maturation can be targeted more efficiently by generating C2C12 cells that stably express shRNA. Limitations of the method are a variability in the knockdown efficiencies, depending on the specific shRNA that may be overcome by using gene knockout strategies based on CRISPR/Cas9, as well as potential off-target effects of the shRNA that should be considered.
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影响因子：
3.7
作者：
Sandri, M;Bortoloso, E;Volpe, P
通讯作者：
Volpe, P
影响因子：
30.8
作者：
Le Goff, Carine;Morice-Picard, Fanny;Dagoneau, Nathalie;Wang, Lauren W.;Perrot, Claire;Crow, Yanick J.;Bauer, Florence;Flori, Elisabeth;Prost-Squarcioni, Catherine;Krakow, Deborah;Ge, Gaoxiang;Greenspan, Daniel S.;Bonnet, Damien;Le Merrer, Martine;Munnich, Arnold;Apte, Suneel S.;Cormier-Daire, Valerie
通讯作者：
Cormier-Daire, Valerie
影响因子：
5.1
作者：
Dodds, E;Dunckley, MG;Dickson, G
通讯作者：
Dickson, G
影响因子：
2.7
作者：
Lawson, MA;Purslow, PP
通讯作者：
Purslow, PP
DOI：
10.1016/j.matbio.2018.03.023
发表时间：
2018-10
期刊：
Matrix biology : journal of the International Society for Matrix Biology
影响因子：
--
作者：
Iozzo RV;Gubbiotti MA
通讯作者：
Gubbiotti MA