摘要
目的利用Tet-On基因表达系统建立Saos-2/Tet-On/TAp73γ细胞株,通过强力霉素(Doxycycline,Dox)定量诱导TAp73γ基因的表达。方法将质粒pRevTet-On、pRevTRE-Luc和pRevTRE-TAp73γ分别转染PT67细胞,产生相应的逆转录病毒。RevTet-On病毒感染Saos-2细胞,经G418选择培养基进行培养并感染RevTRE-Luc,再应用荧光素酶活性检测筛选出高诱导倍数的Saos-2/Tet-On细胞克隆。最后该细胞株感染RevTRE-TAp73γ病毒,用强力霉素筛选出阳性细胞,在不同浓度Dox诱导下用Western blot分析Saos-2/RevTet-On/TAp73γ细胞中TAp73γ的表达。结果Saos-2/RevTet-On克隆5当培养基中Dox浓度达到2·5mg/L时,荧光素酶相对活性值为5·3×106RLU/s,去除Dox时荧光素酶活性最弱,背景最低。Western blot结果显示在Saos-2/Tet-On5细胞中TAp73γ蛋白表达随着Dox浓度的增加而增加。结论建立了Saos-2/Tet-On及Saos-2/Tet-On/TAp73γ细胞株,可利用四环素衍生物定时定量调整Saos-2细胞中TAp73γ基因的表达,为研究TAp73γ细胞内协同作用因子、进一步阐明细胞内p73蛋白的生物学行为提供了一个理想的研究平台。
Objective To established Saos-2/RevTet-On/TAp73γ cell line by using Tet-On gene expression system, and to control the expression of TAp73γ gene in Saos-2 by the induction of doxycycline （Dox） . Methods pRevTet-On, pRevTRE-Luc and pRevTRE-TAp73γ were respectively transfected into package cell line PT67 to produce recombinantion retrovirus. Saos-2 cells were infected by recombinantion retrovirus RevTet-On and selected in the growth medium containing G418. Saos-2/Tet-On cell clone with low background and high induction of luciferase in response to Dox was obtained by the detection of luciferase activity of RevTRE-Luc. Finally, this resulted Saos-2/Tet-On cell was stably infected with recombinant retrovirus RevTRE-TAp73γ and then Western blot analysis was carried out to observe the levels ol TAp73γ expression under the different Dox concentrations. Results Clone 5, one of Saos-2/Tet-On clones, had high lueiferase activity in 2.5mg/L of Dox, and low basal lueiferase expression in the absence of Dox. Western blot showed the TAp73γ expression in clone 5 was increased with the increasing concentration of Dox. Conclusions The RevTet-On gene expression sysytem could be used to modulate the expression TAp73γ in Saos-2 by Dox, providing a useful tool for the further study of the role of cellular TAp73γ.
出处
《肿瘤基础与临床》
2006年第4期273-275,共3页
journal of basic and clinical oncology
