鳜胚胎细胞系的建立与应用

Culture and application of Siniperca chuatsi embryo cell line

为更好地开展鳜基因功能研究和药物筛选工作,提高基因转染效率,本研究以鳜囊胚期胚胎为材料,采用含有20%胎牛血清的DMEM培养基进行培养,建立了生长稳定的鳜胚胎细胞系MFE。在此基础上,采用绿色荧光蛋白(GFP)作为标记物,在HEK293T细胞中体外包装逆转录病毒,再感染MFE细胞系。MTT法分析表明传代后细胞培养96 h内,细胞生长率变化也经历增殖、降低到达稳定期。而且MFE细胞能稳定表达GFP基因,感染效率为20%±5%,而脂质体转染效率为3%±2%。可见包装病毒感染细胞不仅能获得稳转细胞系,效率也远高于脂质体瞬时转染。荧光定量PCR分析表明,MFE细胞系能表达Irf1、Irf3和Irf7基因,Irf1基因表达量最高。MFE细胞系受到poly I:C刺激后,Irf1、Irf3和Irf7的表达量分别升高3.5,2.3和2.1倍。因此,MFE细胞通过病毒感染可以获得较高的转染效率,该细胞可作为鳜免疫相关基因功能研究的工具。

There are only a few cell lines of Siniperca chuatsi(mandarin fish)that could be widely used in scientific research.For mandarin fish,cell culture technique is an important aspect of the quality control program for the National Wild Fish Health Survey.Healthy,sensitive and mycoplasma-free cells are essential for detection of fish viruses in free-ranging fish populations.S.chuatsi is native to China and an important commercial species.In addition,the transfection efficiency is quite low in most fish cell lines.That is an obstacle in the basic research on fishes.In this study,a continuous cell(MFE)culture derived from the mandarin fish embryo was developed and has been subcultured over 20 passages in DMEM cell culture medium.MFE consists predominantly of epitheliallike cells and grows well in DMEM supplemented with 20%fetal bovine serum.Cell proliferation was assessed by MTT assay.The absorbance of the sample was read directly in the wells at an optimal wavelength of 570 nm.The results showed that MFE cell experienced proliferation,decrease and stable phases during 96 h.Meanwhile,we used GFP-expressing retrovirus produced by HEK293T cells to test the transfection efficiency in MFE cell line.The result showed that the transfection efficiency reached 20%±5%without affecting the growth of these cells.Subsequently,the expression of three genes of Irf1,Irf2 and Irf7 was examined in MFE cell line by qRT-PCR.The results indicated that Irf1 is the highest one,and all increased to 3.5,2.3 and 2.1 folds after poly I:C stimulation.The results of this study showed that MFE is the first cell line originated from embryo of mandarin fish.It could be used as a tool for gene function research and genetic modification.