High-Efficiency Lentiviral Gene Modification of Primary Murine Bone-Marrow Mesenchymal Stem Cells
Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However, the efficiency with which they transduce target cells varies significantly, in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning, production, and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene.
Key wordsLentivirus Tangential flow filtration Mesenchymal progenitor cells Protein tyrosine phosphatase receptor type C Stem cell antigen 1 Fluorescence-activated cell sorting Gene modification
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