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本实验应用Tet On基因表达系统 ,通过强力霉素调控血小板生成素 (TPO)基因在CHO细胞中的表达。应用DNA重组技术 ,构建质粒 pTRE TPO。应用脂质体介导的基因转染技术 ,pTRE TPO与pTK Hyg共转染CHO Tet On细胞株 ,得到双稳定细胞株CHO Tet On TPO ,并筛选高表达、低背景克隆。当培养基中不加强力霉素时 ,RT PCR和Western印迹未见TPO表达 ,ELISA测定培养上清TPO水平为 0 .1μg/L。当培养基中加入 2mg/L强力霉素时 ,RT PCR和Western印迹可见TPO表达条带 ,ELISA测定培养上清TPO水平为 10 .8μg/L ,两者相差 10 8倍。本试验通过强力霉素调控TPO基因在CHO细胞中的表达 ,有望为TPO基因治疗提供一条可控的安全途径。
In this experiment, Tet On gene expression system was used to regulate the expression of thrombopoietin (TPO) gene in CHO cells by doxycycline. Plasmid pTRE TPO was constructed using DNA recombination technology. The CHO Tet On cell line was cotransfected with pTRE TPO and pTK Hyg by using liposome-mediated gene transfection technology to obtain the CHO Tet On TPO bovine stable cell line and screened high-expression and low-background clones. When no doxycycline was added in the medium, TPO expression was not detected by RT-PCR and Western blotting. The TPO level in culture supernatant was 0 .1 μg / L by ELISA. When 2 mg / L doxycycline was added to the medium, TPO expression bands were detected by RT PCR and Western blotting. The TPO level in the culture supernatant was 10.8 μg / L by ELISA, which was 108-fold different. In this experiment, the expression of TPO gene in CHO cells through doxycycline is expected to provide a controlled and safe way for TPO gene therapy.