Contributed by Elizabeth Eustis-Turf*, Kiu-Mei Wang, and Lawrence B. Schook, Laboratory of Molecular Immunology, Department of Animal Sciences, University of Illinois, Urbana- Champaign
The transfer of genes into eukaryotic cells permits the study of many things, such as gene regulation, gene structure, and gene function. The ability to insert a gene into a high percentage of hematopoietic stem cells would also make gene therapy a viable option. Our laboratory has been trying to maximize genetic transfer into non-adherent, murine bone marrow cells using electroporation. We have used the Gene Pulser apparatus to electro-transfect either a murine MHC class II gene, ABb, or a plasmid encoding a mutant dihydrofolate reductase enzyme (dhfr) into murine bone marrow cells.1 In these studies, we determined which parameters worked best for transient expression of genes as well as stable integration of genes. Transient expression is one way for us to study gene regulation, but we are also interested in gene therapy, which can only be successful if there is some kind of selection for stable gene integration. Therefore, we varied buffer, capacitance, and voltage in order to define the optimal conditions for both outcomes.
Bone marrow cells were isolated from the femurs of C3H/HeN and C578B1/6
mice, washed, and resuspended in electroporation medium, at 7-9 x 106
cells/ml. Samples of 800 l each were placed in 0.4 cm gap Gene Pulser
cuvettes and 10 l of DNA (1.0 g/ml) added. The cell-DNA suspension was
gently agitated and placed on ice for 15 minutes prior to electroporation.
After receiving the pulse, the samples were placed on ice fo