用PCR和RT-PCR方法对人OSM cDNA转染的小鼠黑色素瘤细胞进行基因组整合和mRNA转录的检定.基因组整合检定时,采用与调控序列和cDNA序列相对应的上、下游引物,以连续的转录单位进行扩增,能够更准确地反映整合与表达的关系;mRNA检定时,采用与cDNA序列和质粒克隆位点与加polyA信号之间序列相对应的上、下游引物,可以区分宿主细胞中内源性与外源性基因的转录.
Genomic integration and mRNA transcripts of human OSM cDNA in transfected mouse melanoma cells were identified by PCR and RT-PCR methods.A sense primer for the regulatory sequence in carrier vector paired with an antisense primer for cDNA was used in integration analysis, a continuous transcription-unit was amplified with the expected size in OSM cDNA transfected cells but not in the wild type or vector control cells, reflecting a more accurate relationship between integration and expression. In transcription analysis, a sense primer for cDNA paired with an antisense primer for a sequence between the multiple cloning site and polyA signal in carrier vector was used to distinguish exogenous transcripts from endogenous gene products. This method is convenient and specific in determining exogenous gene integration and expression in transfectants.
吕星,邢瑞云,孙志贤,裴雪涛,吴祖泽. PCR检定OSM cDNA转染细胞中基因组整合与转录[J].生物化学与生物物理进展,1999,26(5):450-453
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