在BCR/ABL基因组上敲入mRNA不稳定元件可逆转K562细胞恶性转化  

Reversing Malignant Transformation of K562 Cells by Knock in an RNA Destabilizing Element into BCR/ABL Genome

作　　者：刘力[1] 王树蕙[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所

出　　处：《中国生物化学与分子生物学报》2000年第6期703-709,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：北京市自然科学基金! (7992 0 33);卫生部科学研究基金! (98-1-0 0 2 )资助

摘　　要：融合基因 BCR/ABL在慢性粒细胞白血病的恶性转化过程中起着主导作用 .针对融合基因的 3′端构建了一个定点基因打靶质粒 ,p F2 .neo.abl(1 - 4) ,将一段可引发核内 RNA降解的元件 ,URE,定点整合到融合基因 poly(A)位点的上游 .打靶质粒经脂质体转染 K562细胞后 ,在 96孔板上进行 40 0 μg/ml G41 8筛选 ,neor克隆进一步在 2 4孔板上扩增 .以特异性引物经基因组 PCR及Southern印迹分析对阳性克隆进行检测 .研究发现阳性克隆在 96孔板内 3周其增殖状况良好 ,但在 2 4孔板内扩增一周后迅速发生死亡现象 .观察单个阳性克隆在正常培养液增殖情况 ,发现 5d后其细胞周期被完全阻抑 .研究结果说明 ,在转录后期 m RNA水平控制 BCR/ABL融合基因的表达可以抑制慢性粒细胞白血病的恶性转化 .The BCR/ABL fusion gene plays an important role in the malignant transformation of chronic myelogenous leukemia (CML).A gene targeting vector,pF2. neo .abl(1 4) which contains an RNA destabilizing element (URE),was constructed.This vector was designed to integrate to the upstream of the 3′ poly(A) site of the fusion gene through homologous recombination to destabilize BCR/ABL mRNA in cell nucleus.After tranfecting into K562 cells by DMRIE C,individual clone was selected at 400 μl/ml G418 medium in 96 well plates.The neomycin resistant colonies were further expanded in 24 well plates.Positive clones with the correct recombination were confirmed by genomic PCR and Southern blot analysis.Interestingly,after transferring into 24 well plate for a week,the positive clones sharply undergo cell death,although previously they have grown well in 96 well plate for about three weeks,and the cell death progress can be reversed upon the addition of K562 condition medium.A single cell culture from a targeted clone only proliferates for 4-5 days in the normal culture media before the cell proliferation stops.This study indicates that to control BCR/ABL fusion gene expression at the posttranscription level by gene targeting can prevent the malignant transformation of CML.

关 键 词：慢性粒细胞白血病 费城染色体 恶性转化 质粒 

分 类 号：R733.7[医药卫生—肿瘤]