Lan Hai, Zhang Qing-yun, Xu Jian-jun, Wang Ya-ming
Department of Immunology, The School of Oncology, Peking University, Beijing Institute for Cancer Research, Beijing, 100036, China.
Zhonghua Zhong Liu Za Zhi. 2006 May;28(5):337-41.
To verify if mutated polyadenylation signal retroviruses can produce viral-host readthrough transcripts (Rth) and have the ability to transform human gastric epithelial GES-1 cells, and to discuss the new functions of retroviruses in gastric cancer related gene research.
The polyadenylation signal-deficient retrovirus vector mutated by PCR site-directed mutagenesis was used to make polyadenylation signal-deficient retroviruses by PA317 packaging cells. The GES-1 cells were infected by the viruses and selected by G418. Viral-host readthrough RNAs were checked by Northern blot. The cell growth and soft agar assay were run to test the transformed cells.
polyadenylation signal-deficient retroviruses could be packaged by PA317 packaging cells. The viruses had the ability to infect GES-1 cells. Northern blot analysis of viral RNA from infected pools and individual G418-resistant clones demonstrated that mutation of consensus LTR polyadenylation signals generated Rth viral RNA in the infected GES-1 cells. Phenotypic analysis results showed that the GES-1 cells infected with plyadenylation signal mutant viruses tended to grow in a cluster manner. Pools of PA317 cells infected with mutant viruses were able to form colonies in soft agar with a higher efficiency than control or uninfected cells.
Host readthrough transcripts generated by polyadenylation signal mutant viruses may contribute to transformation GES-1 cell phenotypes. The mutant vectors and the method described in the present work may be useful as tools to trap and identify genes involved in retroviral insertion mediated cell transformation.
验证突变的聚腺苷酸化信号逆转录病毒是否能产生病毒-宿主通读转录本(Rth)并具有转化人胃上皮GES-1细胞的能力,并探讨逆转录病毒在胃癌相关基因研究中的新功能。
利用PCR定点诱变突变的聚腺苷酸化信号缺陷型逆转录病毒载体,通过PA317包装细胞制备聚腺苷酸化信号缺陷型逆转录病毒。用该病毒感染GES-1细胞,并用G418进行筛选。通过Northern印迹检测病毒-宿主通读RNA。进行细胞生长和软琼脂试验以检测转化细胞。
PA317包装细胞可包装聚腺苷酸化信号缺陷型逆转录病毒。该病毒具有感染GES-1细胞的能力。对感染细胞池和单个G418抗性克隆的病毒RNA进行Northern印迹分析表明,共有的长末端重复序列(LTR)聚腺苷酸化信号突变在感染的GES-1细胞中产生了Rth病毒RNA。表型分析结果显示,感染聚腺苷酸化信号突变病毒的GES-1细胞倾向于呈簇状生长。感染突变病毒的PA317细胞池能够在软琼脂中形成菌落,且效率高于对照或未感染细胞。
聚腺苷酸化信号突变病毒产生的宿主通读转录本可能有助于转化GES-1细胞表型。本研究中描述的突变载体和方法可能作为捕获和鉴定参与逆转录病毒插入介导的细胞转化的基因的工具。