Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro.
Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro.
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人 T 细胞白血病病毒 1 型癌蛋白在体外抑制 CD34 细胞的多系造血。
DOI：
10.1128/jvi.77.22.12152-12164.2003
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发表时间：
2003
影响因子：
5.4
通讯作者：
Feuer,Gerold
中科院分区：
文献类型：
--
作者：
Tripp,Adam;Liu,Yingxian;Sieburg,Michelle;Montalbano,Joanne;Wrzesinski,Stephen;Feuer,Gerold
材料和方法细胞系将293 T和HeLa细胞在含有10%热灭活胎牛血清（FBS; Gemini，卡拉巴萨斯，CA）的Dulbeccos改良Eagle培养基（DMEM）（Gibco-BRL，Grand Island，NY）中培养，2 mM L-谷氨酰胺（Gibco-BRL），100 U青霉素/ml和100 μ g链霉素/ml（Pen/Strep; Gemini）。Jurkat和CEM细胞系在Iscove改良的Dulbecco培养基（IMDM）（Gibco-BRL）中培养，该培养基补充有10%FBS、2 mM L-谷氨酰胺和Pen/Strep，在37 ℃下，在具有5%CO2的潮湿培养箱中。Tax反式激活测定。将慢病毒载体构建体与pHTLV-1-LTR-CAT或pHTLV-2-LTR-CAT共转染到293 T细胞中，所述构建体分别编码从HTLV-1 LTR或HTLV-2 LTR表达的细菌氯霉素乙酰转移酶（CAT）基因。然后，通过使用磷酸钙DNA沉淀法（38），将2.5g每种慢病毒载体构建体与2.5g pHTLV-1-LTR-CAT或pHTLV-2-LTR-CAT共转染到293 T细胞（5.0 × 105）中。通过使用冻融方法裂解细胞沉淀，并分析5g细胞提取物的CAT活性。通过使用Bradford测定法测试细胞提取物，并且如前所述（8），针对蛋白质的量对CAT反应进行标准化。通过使用分子动力学（Molecular Dynamics）（桑尼维尔，加利福尼亚州）测量乙酰化产物。PhosphorImager 445 SI，然后通过使用Molecular Dynamics ImageQuant 5.1程序分析CAT活性。如前所述构建pHRCMV-Tax 1/GFP和pHRCMVTax 1（）/GFP（97）。通过PCR扩增从BC 20分离HTLV-2 tax cDNA。2（来自Irvin S. Chen，加州大学，洛杉矶）（42），通过使用正向引物25-mer（5-TGCGCTCGAGACCACCAACACCATG-3）和反向引物25-mer（5-TGGGATCCCTAGTCGCCATTGTCAT-3）。将扩增的cDNA片段亚克隆到PCR克隆载体pCR 2中。1（Invitrogen，卡尔斯巴德，加利福尼亚州），产生pCR 2。1税收2.通过pCR 2的BamHI和XhoI限制性消化分离Tax 2片段。将重组质粒pHRCMV-Tax 1/GFP连接到pHRCMV-Tax 2载体上，用XhoI和BamHI双酶切，分离出tax 1基因，凝胶电泳纯化。pHRCMV-Tax 2/GFP的测序证实了tax 2基因的插入。pHRCMV-Vpr/GFP是来自
MATERIALS AND METHODSCell lines. 293T and HeLa cells were cultured in Dulbeccos modified Eagle medium (DMEM)(Gibco-BRL, Grand Island, NY) with 10% heat-inactivated fetal bovine serum (FBS; Gemini, Calabasas, Calif.), 2 mM L-glutamine (Gibco-BRL), and 100 U of penicillin/ml and 100 g of streptomycin/ml (Pen/Strep; Gemini). Jurkat and CEM cell lines were cultured in Iscove modified Dulbecco medium (IMDM)(Gibco-BRL), supplemented with 10% FBS, 2 mM L-glutamine, and Pen/Strep at 37 C in a humidified incubator with 5% CO2. Tax transactivation assay. Lentiviral vector constructs were cotransfected with pHTLV-1-LTR-CAT or pHTLV-2-LTR-CAT into 293T cells, constructs encoding the bacterial chloramphenicol acetyltransferase (CAT) gene expressed from the HTLV-1 LTR or HTLV-2 LTR, respectively. Then, 2.5 g of each lentiviral vector construct was cotransfected with 2.5 g of pHTLV-1-LTR-CAT or pHTLV-2-LTR-CAT into 293T cells (5.0 105) by using the calcium phosphate DNA precipitation method (38). Cell pellets were lysed by using the freeze-thaw method, and 5 g of the cell extract was analyzed for CAT activity. Cell extracts were tested by using a Bradford assay, and CAT reactions were normalized for the amounts of protein, as previously described (8). Acetylated products were measured by using a Molecular Dynamics (Sunnyvale, Calif.) PhosphorImager 445SI and then analyzed for CAT activity by using the Molecular Dynamics ImageQuant 5.1 program.Lentiviral vector construction. The pHRCMV-Tax1/GFP and pHRCMVTax1 ()/GFP were constructed as previously described (97). The HTLV-2 tax cDNA was isolated by PCR amplification from BC20. 2 (a gift from Irvin S. Chen, University of California, Los Angeles)(42), by using forward primer 25-mer (5-TGCGCTCGAGACCACCAACACCATG-3) and reverse primer 25-mer (5-TGGGATCCCTAGTCGCCATTGTCAT-3). The amplified cDNA fragment was subcloned into the PCR cloning vector pCR2. 1 (Invitrogen, Carlsbad, Calif.), creating pCR2. 1Tax2. The Tax2 fragment was isolated by BamHI and XhoI restriction digestion of pCR2. 1Tax2 and ligated into pHRCMV-Tax1/GFP, which was first digested with XhoI and BamHI to liberate the tax1 gene and then purified by gel electrophoresis. Sequencing of pHRCMV-Tax2/GFP confirmed the insertion of the tax2 gene. pHRCMV-Vpr/GFP was a gift from