【摘要】 为了识别参与细胞癌变的基因,本文选择p53^135-val-过表达正常细胞系R6#13-8及其自发转化癌变细胞系T2作为研究材料,在克隆差异表达基因的同时,建立了一种简单、快速的差异表达基因分离的方法,并已克隆到2个与细胞癌变相关的候选新基因。本文所得的实验结果表明:这种方法实验步骤简单,避免使用同位素,RT-PCP扩增带的重复性好,能克隆到大于500bp的差异表达cDNA片段,差异表达的PCR cDNA片段假阳性率低,并可广泛适用于在两个或两个以上相对应真核细胞RNA群体中分离和克隆特异表达基因。研究结果还提示:在R6#13-8自发转化为T2的过程中涉及多个基因的激活与失活,这两个细胞系之间存在明显的基因表达差异。更多还原

【Abstract】 The p53 gene is the most frequently mutated gene identified so far in human cancers. When a mutant p53135-val gene was allowed to be over-expressed in Rat6(R6) cells, a high incidence of spontaneous transformation was observed in long-term culture of this cell line(R6 # 13-8). To identify genes involved in cell transformation, parental p53 over-expressing cell, R6#13-8, and its spontaneous transformant T2, were analyzed by an improved mRNA differential display technique, which was reproducible, simpler, and was able to clone cDNA longer than 500bp, and was with less false positives.When 33 10-mer or 12-mer single primerswith arbitrary but defined sequence were used for PCR, over 90 discrete cDNAs were obtained from R6# 13-8 and T2 cells. Three differentially expressed cDNAs were identified, one of them is highly expressed in T2 cells, while the other two, 0. 8kb and 0. 9kb long, are highly expressed in R6 # 13-8 cells. The latter were cloned and confirmed by Northern hybridization. Both cloned fragment were not homologous with any published sequence.Our results suggest that the activation and inactivation of genes are involved in the process of the spontaneous transformation from R6# 13-8 to T2.更多还原