FBXO6稳定表达肿瘤细胞系的构建及其亚细胞器定位

检测FBXO6在多种肿瘤细胞中的表达和定位情况并构建稳定表达FBXO6的肿瘤细胞株。运用RT-PCR方法,在人胚肾293T细胞以及9种不同来源的肿瘤细胞中,检测了FBXO6的表达情况。以载体质粒pBabe-3Flag-FBXO6-puro/pBabe-3Flag-con、包装质粒pCMV-GAG-POL及包膜质粒pCMV-VSVG用Lipofectamine2000共转染包装细胞系293T,收集病毒颗粒感染A549细胞,经嘌呤霉素筛选稳定表达细胞株,Western blot鉴定FBXO6的表达。利用激光共聚焦荧光显微镜,采用间接免疫荧光方法,检测外源性FBXO6在A549中的亚细胞定位。在10种不同来源的细胞株中,FBXO6在A549细胞中的表达最高。成功筛选出嘌呤霉素抗性细胞系A549-Con与A549-3Flag-FBXO6。Western blot方法发现A549-3Flag-FBXO6细胞系稳定表达FBXO6蛋白。间接免疫荧光发现FBXO6与内质网tracker有共定位。FBXO6在肺癌A549细胞中高度表达,构建了稳定表达FBXO6的A549细胞系,部分FBXO6分布在内质网中。

The construction of cancer celllines stably expressing FBXO6 and its subcellularlocalization in A549 cells

The expression and localization of FBXO6 in a variety of tumor cells has been detected,and a tumor cell line stably expressing FBXO6 has been constructed in this paper.The expression of FBXO6 was examined by RT-PCR method in both human embryonic kidney 293T cells,and nine kinds of tumor cells from different sources.Packaging cell line 293T was co-transfected with pBabe-3Flag-FBXO6-puro/pBabe-3Flag-con,packaging plasmid pCMV-GAG-POL and envelope plasmid pCMV-VSVG by Lipofectamine2000.Virus particles were collected and A549 cells were infected.Cells were selected by puromycin to get stably expressing strains.Western blot was used to detect FBXO6 expression.Laser confocal fluorescence microscopy and indirect immunofluorescence were used to detect the subcellular localization of exogenous FBXO6 in A549 cells.A549 cells expressed the highest FBXO6 among the 10 different sources of cell lines.Puromycin-resistant cell lines A549-Con and A549-3Flag-FBXO6 were selected.A549-3Flag-FBXO6 stably expressing FBXO6 protein was confirmed by Western blot.Indirect immunofluores-cence found that FBXO6 was co-localized with endoplasmic reticulum tracker.FBXO6 was highly expressed in lung cancer A549 cells.A549 cells stably expressing FBXO6 were constructed.FBXO6 was partially distributed in the endoplasmic reticulum.