Previous attempts in using recombinant viral vectors to transfer genes into bone marrow cells have resulted in expression frequencies of 2-15% (8, 9, 12, 13, 14, 24). These vectors, however, require complex gene vector constructions and culture methods and have been difficult to utilize. We therefore have used electroporation to establish a less complicated and equally efficient technique for gene transfer. Conditions yielding high gene transfer into bone marrow precursor cells by electroporation were determined using a mutant dihydrofolate reductase gene and/or murine MHC class II genes. Successful transfer and expression of these genes were assessed by (1) fluorescence staining using monoclonal antibodies, (2) acquired resistance to the anti-folate drug methotrexate and (3) Southern blot analysis. Stable integration of the newly acquired mutant dihydrofolate reductase gene was observed in > 25% of murine hematopoietic progenitor cells (CFU-M). Electroporation of class II genes in conjunction with the mutant dihydrofolate reductase and subsequent selection with methotrexate resulted in expression of transfected Ia molecules in 12-15% of bone marrow derived macrophages. Integration of the transferred major histocompatibility genes was verified by Southern blot analysis of high molecular weight cellular DNA following electroporation and methotrexate selection. The frequencies of gene transfer and expression achieved in this study suggest that electroporation is a viable option for monitoring gene expression in bone marrow derived cells.
ASJC Scopus subject areas
- Animal Science and Zoology