off调控bcr-abl基因表达细胞系 293pT2P210建立.docVIP

off调控bcr/abl基因表达细胞系 293pT2P210建立 　作者：黄文荣 鲁茁壮 王立生 王华　段海峰 李庆芳 高春记 达万明 【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293 pT2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。 【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 论文代写 　　 Construction of 293pT2P210 Cell Line Enables Expression of bcr/abl to be Regulated by Tetoff InducingExpressionSyste Abstract Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210bcr/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 induci