GAL enzyme. Subsequent oxidization and self-coupling forms an 
indigo blue derivative. The vector containing cells can thus be 
discriminated from unmodified cells and then be enumerated with 
light microscopy. Rats were inoculated with 4xl0 4 9L gliosarcoma 
cells into the right cerebral hemisphere by stereotaxic guidance. 
Seven days later, 3xl0 6 PA317/GlBgSvNa VPC were injected into the 
tumor bearing and non-tumor bearing hemispheres using the same 
stereotaxic coordinates. Five, 9, and 15 days after injection of 
the VPC, the rats were sacrificed by an intracardiac injection of 
formaldehyde to fix the brain. The brains were stained with X- 
GAL. Control rats received GlBgSvNa.29 transduced, G418 selected 
3T3 non-producer cells. The VPC and the control GlBgSvNa.29 
transduced 3T3 cells were 100% positive by X-GAL staining prior to 
injection. In this experiment, we have shown that the injection 
of VPC led to transduction of 10-55% of the tumor cells in situ . 
Figure 2 (Appendix A: Reprint 2) presents the microscopic sections 
taken from the rats on day 7 and 14. The injected VPC disappeared 
from the injection site after day 14. There was a clear cut 
delineation between the transduced tumor cells and normal brain 
tissue. Except for the possibility of rare transduced endothelial 
cell in the vicinity of the tumor, no evidence of non-tumor brain 
transduction was evident in either cerebral hemisphere as shown in 
Figure 3 (Appendix A: Reprint 2) . The most mitotically active 
endothelial cells in the area of the tumor are likely to be those 
responding to neovascularization signal within the tumor. 
Destruction of transduced endothelial cells with GCV therapy within 
the tumor may enhance the anti-tumor activity. The non-producer 
control cells did not demonstrate transduction of either brain and 
they were not seen in the tumor or brain after 14 days. This 
experiment demonstrates that local injection of a VPC 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 
and that the producer cells disappear after 14 days. 
B. In Vitro GCV Sensitivity of Mammalian Cells +/- Transduction with 
a HS-tk Vector 
The GlTkSvNa.29 vector was transduced into the cell lines 
listed in Table 1, Appendix E in vitro using filtered supernatant 
from confluent producer cells. The transduced cell lines were then 
selected in G418 for 7 days at 1.0 mg/ml. Only cells expressing 
a functional NeoR gene are able to survive these G418 culture 
conditions producing an essentially 100% selected population of 
vector transduced cells. We then evaluated the sensitivity of the 
transduced G418-selected cells compared to the non-transduced 
parent cell lines. In each case, the HS-tk transduced, G418 
selected cell lines were markedly more sensitive to low 
concentrations of GCV. Concentrations of 0.5-5 ug/ml were 
inhibitory to the HS-tk transduced cells in this assay. In a 
clonogenic assay, HS-tk positive cells were completely inhibited 
at 0.5 ug/ml. These findings confirm that HS-tk gene-containing 
retroviral vectors can effectively transduce murine and rat tumor 
cells and stably express both the NeoR and HS-tk genes resulting 
in 100% kill of the transduced cells in vitro when exposed to GCV. 
Recombinant DNA Research, Volume 18 
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