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本文总结应用DNA-磷酸钙盐沉淀的基因转移技术,把含人c-sis cDNA的质粒pSM-1,以单独转染或与pSV_2neo共转染方法转入CHO细胞(中国仓鼠卵巢细胞),经低血清或G_(418)筛选分离得到多个有PDGF表达的细胞株。其中PDGF高表达件——FB_6,细胞形态和生长行为明显改变,可在软琼脂培基上形成集落,细胞生长速率加快,能在低血清(2%)培液中长期传代,细胞的条件培液有刺激静止的NRK细胞(大鼠肾成纤维细胞)DNA合成的活力。RNA点杂交和Southern Blot显示FB_5细胞有??PDGF mRNA的高表达和人c-sis基因的整合,而且,在连续传代7个月后,FB_5细胞基因组中仍然有人c-sis基因存在,说明CHO细胞的不正常生长和转化是由于PDGF基因的稳定整合和高表达所引起。这一稳定转化株(FB_5)是进一步研究PDGF对细胞生长控制和转化功能的理想模型。
This article summarizes the gene transfer technology using DNA-calcium phosphate precipitation. The plasmid pSM-1 containing human c-sis cDNA is transfected into CHO cells (Chinese hamster ovary cells) either alone or co-transfected with pSV_2neo. Low serum or G_ (418) screening to obtain a plurality of PDGF-expressing cell lines. Among them, the expression of FBG6, the cell morphology and the growth behavior of PDGF, significantly changed. Colonies could be formed on soft agar, the cell growth rate was accelerated, and the cells could be passaged in low serum (2%) medium for long term. Fluid stimulates the synthesis of DNA in resting NRK cells (rat kidney fibroblasts). RNA dot blot and Southern Blot showed that FB_5 cells had high expression of PDGF mRNA and integration of human c-sis gene. Moreover, there was still human c-sis gene in the genome of FB_5 cells after 7 months of continuous passage, indicating that CHO Aberrant cell growth and transformation is due to the stable integration and high expression of the PDGF gene. This stable transformant (FB_5) is an ideal model to further study the function of PDGF on cell growth control and transformation.