TEC启动子介导BCR/ABL基因在BaF3细胞中的表达  

TEC Promoter Mediates P210^(bcr/abl)Gene Expression in BaF3 Cells

作　　者：朱玉峰[1] 王元占[1] 孟凡义[2] 

机构地区：[1]南方医科大学南方医院实验动物研究中心,广东广州510515 [2]南方医科大学南方医院血液病科,广东广州510515

出　　处：《中国实验血液学杂志》2012年第3期769-772,共4页Journal of Experimental Hematology

基　　金：广东省科技计划项目资助;编号2005B60302005

摘　　要：转P210bcr/abl基因小鼠是研究慢性髓系白血病的理想模型,但广泛表达的启动子容易引起转基因小鼠的胚胎致死,所以应用在造血细胞中特异表达的启动子是成功建立转P210bcr/abl基因小鼠模型的关键。本研究探讨TEC启动子介导BCR/ABL基因在BF3细胞中的表达。从小鼠的基因组中克隆TEC启动子的-364至+22区域,并替换掉IRES2-eGFP载体的CMVie启动子,同时在该载体的EcorⅠ酶切位点插入7.2 kb的P210bcr/abl基因,再将构建好的载体分别转染BaF3细胞和293细胞,观察eGFP基因与P210bcr/abl基因在2种细胞中的表达情况。结果通过荧光显微镜和流式细胞仪检测发现,eGFP基因在BaF3细胞成功表达,表达率在转染后6、24、72 h分别为7.10%、23.35%和64.61%,而在293细胞中未见eG FP基因表达。RT-PCR在BaF3细胞中扩增出BCR/ABL mRNA中372 bp的片段,而在293细胞中未扩增出任何片段。结论:TEC启动子的-364至+22区域能介导相关基因在造血细胞中高效特异性表达,适合于构建转P210bcr/abl基因小鼠模型。P210bcr/abl transgene mouse is a good model to reaearch the chronic myelogenous leukemia（CML）,but the P210bcr/abl gene has a lethal effect on embryogenesis if driven by the constitutive promoter.So,the use of promoter which induces the special expression in hematopoietic tissue is the key to construct CML transgenic mice.This study was purposed to investigate the TEC promoter mediated P210bcr/abl gene expression in BaF3 cells.The CMVie promotes of IRES2-eGFP vector was replaced with the-364—＋22 domain of TEC promoter cloned from mouse genome,and the P210bcr/abl gene was inserted into the EcoR Ⅰ site of TEC-IRES2-eGFP vector.Then,the constructed vector was transfected into the BaF3 cells and 293 cells respectively.The expression levels of eGFP gene and P210bcr/abl gene in BaF3 and 293 cells were detected.The results showed that with fluorescent microscopy and flow cytometry,the eGFP gene was found to be expressed in the BaF3 cells,the expression rate was 7.10%,23.35%,64.61% at 6,24,72 h respectively after transfection,but the fluorescence was not seen in 293 cells.A 372 bp fragment of BCR/ABL mRNA was amplified by RT-PCR in BaF3 cells,but not in 293 cells.It is concluded that the-364—＋22 domain of TEC promoter can mediate high-effective and specific expression of related genes in hematopoietic tissue,which can be used to construct P210bcr/abl transgene mice model.

关 键 词：TEC启动子 BCR/ABL基因 BaF3细胞 转基因小鼠模型 

分 类 号：R332[医药卫生—人体生理学] R733.72[医药卫生—基础医学]