Tet-off调控bcr/abl基因表达的细胞系293pT2-P210的建立  

Construction of 293pT2-P210 Cell Line Enables Expression of bcr/abl to be Regulated by Tet-off Inducing-Expression-System

作　　者：黄文荣[1] 鲁茁壮[2] 王立生[2] 王华[2] 段海峰[2] 李庆芳[2] 高春记[1] 达万明[1] 

机构地区：[1]解放军总医院血液科,北京100853 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中国实验血液学杂志》2007年第2期224-228,共5页Journal of Experimental Hematology

摘　　要：慢性髓系白血病(CML)是一种以髓系细胞受累为主的克隆性疾病。目前认为CML的主要分子发病基础为:位于9q34的abl基因与位于22q11的bcr基因相互易位形成bcr/abl融合基因,并编码具有很强蛋白酪氨酸激酶活性的P210bcr/abl融合蛋白,该蛋白通过多种信号通路抑制细胞凋亡而导致细胞转化。本研究为构建能由Tet-off诱导表达系统调控bcr/abl融合基因表达的细胞系,为深入研究bcr/abl融合基因功能及其调控机制提供一种有价值的细胞模型。将bcr/abl融合基因亚克隆到pTRE2hyg载体构建重组质粒pT2-P210,同时将Tet-off质粒转染293细胞并筛选出单克隆,然后进一步转染pTRE2hyg-LUC质粒,通过荧光素酶活性检测选出能有效诱导目的基因表达的293Tet-off细胞,然后将重组质粒pT2-P210转染到293Tet-off细胞中。结果表明:筛选出了bcr/abl融合基因能被强力霉素有效调控的293pT2-P210单克隆细胞。结论:本研究筛选的293pT2-P210细胞为bcr/abl基因表达能被Tet-off诱导表达系统调控的细胞系,它们适用于bcr/abl融合基因功能及其信号调控机制的深入研究。Chronic myelogenous leukemia （CML） is a clonal myeloproliferative disease of transformed hematopoietic progenitor ceils. It is now clear that the chimeric bcr/abl P210^bcr/abl fusion protein, which is generated by the reciprocal translocation t（9;22）, inhibits apoptosis and increase proliferation. P210bcr/abl plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable to thoroughly study the function of bcr/abl fusion gene and its signal regulation mechanisin.

关 键 词：BCR/ABL融合基因 Tet-off诱导表达系统 293pT2-P210细胞系 293细胞 

分 类 号：R733.72[医药卫生—肿瘤]