2.d 
This experiment demonstrates that local injection of a vector producer cell 
line will transduce tumor cells in vivo, that the transduced tumor infiltrates surrounding 
brain with the non-transduced tumor and that in vivo retroviral-mediated gene transfer 
does not significantly effect adjacent normal non-proliferating brain tissue. 
B. In Vitro GCV Sensitivity of Mammalian Cells +/- Transduction with a HS-tk 
Vector 
The NeoR-HS-tk vector was inserted into murine (NIH 3T3 cells and the 
MCA 205 fibrosarcoma), rat (9L gliosarcoma), and human (U251 glioblastoma) cell 
lines in vitro using supernate collected from confluent producer line cells. The 
transduced cell lines were then selected in G418 for 14 days at 1.0 mg/ml. Only cells 
expressing a functional NeoR gene are able to survive these G418 culture conditions 
producing a 100% selected population of vector containing cells. 
We then evaluated the sensitivity of the transduced, G418-selected cells 
compared to the non-transduced parent cell lines (figure 3). In each case, the HS-tk- 
transduced, G418 selected 3T3 cell lines and HS-tk producer cell line were markedly 
more sensitive to low concentrations of GCV. Concentrations of 0.5-5 pg/ml were 
inhibitory to the HS-tk-transduced cells in this assay. In a clonogenic assay, HS-tk 
Recombinant DNA Research, Volume 15 
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