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逆转录病毒介导重组CD23 cDNA 稳定整合的RVc23 |
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宋 辉 晋康新 王卫莉 唐 莉 刘 芳 徐德胜
摘 要 目的：建立稳定整合的细胞株，为研究外源基因定位整合机制及其对靶细胞生物特征影响，建立一个生物模型。方法：构建重组病毒载体PLXCD；用Lipofectin将PLXCD转染PA317包装细胞，用G418筛选获得抗性细胞克隆，扩增抗性细胞收集病毒上清；测定病毒上清效价，感染RPMI8866细胞，用G418筛选获得抗性细胞(RVc23-8866细胞)；用FACS分析RVc23-8866细胞膜蛋白分子CD23的表达；用细胞原位杂交(FISH)分析细胞染色体基因组的改变。结果：建立了稳定整合的RVc23-8866细胞株。结论：FISH结果表明PLXCD重组体已整合到细胞基因组中；FACS分析表明RVc23-8866细胞膜表面CD23分子的表达量明显降低，并说明了整合入基因组的CD23片段影响了固有CD23基因的表达。
关键词 逆转录病毒 B淋巴细胞 CD23 RVc23-8866
中国图书分类号 R392.12
Establishment of RVc23-8866 cell line transfected by retrovirus mediated recombinant CD23 cDNA
SONG Hui, JIN Kang-Xin,WANG Wei-Li et al.
Department of Cell Biology,Beijing Medical University,Beijing 100083
Abstract Objective:To establish a stable cell line and employ the cell line as a model to study target integration of exogenous cDNA and modulation of gene expression.Methods:Establish recombinant retrovirus vector PLXCD;use PLXCD to transfect PA317 cells by lipofectin,screen the transfected cells by G418,culture the cells to get supernatant containing viruses;measure the titer of the virus-containing supernatant and transfect RPMI-8866 cells,screen the RVc23-8866 cells by G418;analyze the expression of CD23 on the membrane of RVc23-8866 by FACS;analyze the change of genome by In Situ Hybridization (FISH Method).Results:A stable cell line,RVc23-8866 was established.Conclusions:The results of FISH indicate that the recombinant PLXCD has integrated into the genome of RVc23-8866 cells;the results of FACS indicate that the expression of RVc23-8866 cells;the results of FACS indicate that the expression of membranal CD23 diminished and the integrated CD23 cDNA fragment has effected the expression of inherent CD23 gene.
Key words Retrovirus B lymphocyte CD23 RVc23-8866
随着分子生物学与分子遗传学的迅速发展和研究深入，使得核酸作为一种生物治疗手段正逐步成为现实。但是在实际应用中尚有许多问题没有得到解决，这在某种程度上限制它的广泛应用。CD23主要是分布在B细胞膜上的45 kD的蛋白分子，具有许多生物功能，在B细胞上发挥着重要的作用［1-3］。为了解决基因治疗中最为关键的外源基因靶向定[1] [2] 下一页
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