( r a second 15 minute incubat...)Electroporation of Primary Bone Marrow Cells

r a second 15 minute incubation. Control cultures consisting of cells but no DNA were treated exactly like the test samples. After the second incubation on ice, the control and test cells were cultured in liquid culture in the present of CSF-1. The transient expression of the class II gene was monitored by staining the cells with the appropriate monoclonal antibody (BP 107) and analysis on a Coulter Epics C cytofluorometer. The stable integration of the dhfr gene was demonstrated by the acquired ability of cells to survive in selective medium containing the anti-folate drug, methotrexate (MTX). For colony-forming unit (CFU) assays, 0.33% noble agar was added to the medium, and the number of colonies was counted after 10 days in culture. Cells receiving the dhfr plasmid were cultured with increasing amounts of MTX, from 0-500 nM. Genomic DNA was isolated from the bone marrow cells for Southern blot analysis.^2,3

As shown in Table 1, a 3.5-fold increase was seen in the number of bone marrow stem cells able to survive in 500 nM MTX, when 500 F and 300 V (750 V/cm) Gene Pulser settings were used and a pulse time constant of 10 msec was achieved. Southern blot analysis was done to verify the presence of the dhfr gene within the cultured cells. High molecular weight genomic DNA was isolated from cells that had been pulsed seven days previously and cultured in MTX. Figure 1 reveals hybridization of the dhfr-containing plasmid to sequences within the test cells, yet no hybridization to control cell DNA. The presence of the dhfr gene within the test cells, together with the evidence of expression of that gene due to survival in MTX, indicate the stable integration of the pulsed DNA into the bone marrow ge
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