High-efficient genetic transfection of CD41~+,UT7,U937 and MDA-MB-435 cells with a recombined murine stem cell retroviral vector / 中国病理生理杂志

ABSTRACT

AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector EC1-4 gene (repeats 1-4 of cadherin-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41 + cells and cell culture Cells expressing CD34 + from cord blood were isolated. The inducement of cells expressing CD41 from CD34 + cells was performed by using TPO and cells CD41 + were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot Transduced CD41 +,UT7,U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1?10 7) and EC1-4 pMSCV retroviruses (1.0?10 8). With 8-folds dilution retroviruses,60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells,72.56% in U937 cells and 30.57% in CD41 + cells,respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41 + and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION: The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41 +,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.