Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches.
Genetic manipulation of myoblasts and a novel primary myosatellite cell culture system: comparing and optimizing approaches.
复制标题
DOI：
10.1111/febs.12072
复制
发表时间：
2013-02
期刊：
影响因子：
--
通讯作者：
Rodgers BD
中科院分区：
文献类型：
--
作者：
Jackson MF;Hoversten KE;Powers JM;Trobridge GD;Rodgers BD
体外骨骼肌细胞的遗传操作是众所周知的困难，特别是当使用未分化的肌肉细胞系（成肌细胞）或原代肌肉干细胞（肌卫星）时。因此，我们优化了基因转移的方法过表达绿色荧光蛋白（GFP）在小鼠C2 C12细胞和一个新的系统，主要虹鳟鱼肌卫星细胞。使用常见的基于脂质的转染试剂（Lipofectamine 2000）沿着三种不同的病毒载体：腺相关病毒血清型2（AAV 2）、杆状病毒（BAC）和慢病毒。在优化细胞密度和试剂：DNA比例后，在C2 C12细胞中获得了49%的最大转染效率，尽管GFP信号随着增殖而迅速消散，并且不随着分化而维持。通过延长孵育时间和降低细胞密度，将AAV 2的转导效率优化至65%，尽管通过后仅30%的细胞保留表达。病毒比较显示，慢病毒在转导C2 C12成肌细胞方面最有效，因为97%的细胞仅用106个病毒基因组（vg）转导，而用108 vg AAV 2转导的细胞比例为54%，用109 vg BAC转导的细胞比例为23%。慢病毒还转导90%的初级鳟鱼肌卫星，相比之下，用AAV 2和BAC转导1-10%。在C2 C12细胞中，磷酸甘油酸激酶1启动子的活性是巨细胞病毒立即早期启动子的10倍，两者在鳟鱼肌卫星中均有效。通过分化先前用慢病毒和pgk启动子转导的成肌细胞来实现C2 C12肌管的最大转导。因此，我们的优化方案在不同的肌肉细胞系统中证明是非常有效的，因此可以帮助克服常见的技术障碍。
The genetic manipulation of skeletal muscle cells in vitro is notoriously difficult, especially when using undifferentiated muscle cell lines (myoblasts) or primary muscle stem cells (myosatellites). We therefore optimized methods of gene transfer by overexpressing green fluorescent protein (GFP) in mouse C2C12 cells and in a novel system, primary rainbow trout myosatellite cells. A common lipid-based transfection reagent was used (Lipofectamine 2000) along with three different viral vectors: adeno-associated virus serotype 2 (AAV2), baculovirus (BAC) and lentivirus. Maximal transfection efficiencies of 49% were obtained in C2C12 cells after optimizing cell density and reagent:DNA ratio, although GFP signal rapidly dissipated with proliferation and was not maintained with differentiation. The transduction efficiency of AAV2 was optimized to 65% by extending incubation time and decreasing cell density, although only 30% of cells retained expression after passing. A viral comparison revealed that lentivirus was most efficient at transducing C2C12 myoblasts as 97% of cells were transduced with only 106 viral genomes (vg) compared to 54% with 108 vg AAV2 and 23% with 109 vg BAC. Lentivirus also transduced 90% of primary trout myosatellites compared to 1–10% with AAV2 and BAC. The phosphoglycerate kinase 1 promoter was 10-fold more active than the cytomegalovirus immediate-early promoter in C2C12 cells and both were effective in trout myosatellites. Maximal transduction of C2C12 myotubes was achieved by differentiating myoblasts previously transduced with lentivirus and the pgk promoter. Thus, our optimized protocol proved highly effective in diverse muscle cell systems and could therefore help overcome a common technological barrier.
登录
查看更多内容
影响因子：
14.9
作者：
Kreiss, P;Cameron, B;Pitard, B
通讯作者：
Pitard, B
影响因子：
5.1
作者：
Dodds, E;Dunckley, MG;Dickson, G
通讯作者：
Dickson, G
影响因子：
5.4
作者：
Ferrari, FK;Samulski, T;Samulski, RJ
通讯作者：
Samulski, RJ
影响因子：
5.4
作者：
Dull, T;Zufferey, R;Naldini, L
通讯作者：
Naldini, L
影响因子：
3.7
作者：
Dion, LD;Goldsmith, KT;Garver, RI
通讯作者：
Garver, RI