一种可高效转染的青鳉肌肉细胞系的建立与应用(英文)

摘要

在哺乳动物细胞系中转基因的效率相对较高而在鱼类细胞系中相对较低, 为了获得鱼类高效转基因细胞系, 我们从模式鱼类青鳉(Oryzias latipes)中分离和构建了肌肉细胞系OLM.该细胞系类似于纤维细胞, 在 28℃ DMEM和10％的胚胎牛血清中培养了超过88代.通过染色体分析, 它属于两倍体并含有48条染色体.在脂质体介导下, 报告基因的转染效率可以达到40％, 在测试的其他鱼类细胞中转染效率最高.此外还构建了稳定转染转基因或者非编码RNA的细胞系.OLM细胞对常见的病毒SVCV、GCRV、SGIV等不敏感.综上所述, 研究在青鳉中建立了一种能高效转染的肌肉细胞系, 它适用于鱼类瞬时和稳定的转基因研究.%Transgenic efficiency in mammal cell lines is usually higher but relatively lower in the fish cell lines. Therefore, establishment of any fish cell lines with high transfection efficiency is needed, ideally in the fish model such as medaka (Oryzias latipes). In the present study, we derive a new cell line from medaka muscle termed as OLM (Oryzias latipes muscle), which was maintained in DMEM/HEPES supplemented with 10％ bovine serum at 28℃. Up to now, OLM cells have been subcultured over 88 passages and mainly exhibited a fibroblast-like morphology. By chromosomal analysis, OLM has a modal diploid chromosome number of 48. Moreover, transient transfection efficiency by liposome of OLM cell line is up to 40％, which is much higher than that of the common fish cell lines as tested. In addition, the stable OLMs expressing exogenic non-coding RNA or gene are established. Unexpectedly, the OLM is resistant to the fish virus such as Spring viraemia of carp viruses (SVCV), Grass carp reovirus (GCRV) and Singapore grouper iridovirus (SGIV). Taken together, we have established a stable muscle cell line from model fish medaka as an in vitro model for transgenic application.