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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。
In order to identify the genes involved in the carcinogenesis of cancer cells, we selected R6 ~ 13-8, a normal cell line with p53 ~ (135-val) overexpression, and its spontaneous transformed cancer cell line T2 as the research material, cloned the differentially expressed genes A simple and rapid method for differentially expressed genes and cloned into two candidate new genes related to cell carcinogenesis. The experimental results obtained in this paper show that this method has simple experimental steps and avoids the use of isotopes. The reproducibility of the RT-PCR amplified band is good, and the differentially expressed cDNA fragments larger than 500 bp can be cloned. The false positive rate of differentially expressed PCR cDNA fragments is low , And is widely applicable to the isolation and cloning of specifically expressed genes in two or more corresponding eukaryotic RNA populations. The results also suggest that the activation and inactivation of multiple genes involved in the spontaneous conversion of R6 # 13-8 to T2, with significant differences in gene expression between these two cell lines.