【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。更多还原

【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.更多还原