below this threshold coupled with GVC should be toxic to dividing tumor cells while sparing 
non-dividing CNS cellular constituents. 
MRI Analysis of Baboon Brains Treated with Moderate and High Doses of ADV/RSV-tk 
The gross neuropathological alterations appear to be reflected in the MRI findings. 
Unfortunately, our high dose vector plus GCV animals succumbed before follow-up MRI's 
could be obtained. The high dose vector without GCV animals both exhibited areas of high 
signal intensity on T2 weighted images at three weeks corresponding to the cystic cavities 
seen at three and six weeks at necropsy. At three weeks some minimal mass effect was 
demonstrable by MRI. The six week MRI of the remaining animal in this group showed 
better delineation of the cystic cavity corresponding to the gross pathological finding of a 
resolving circumscribed cavity. Leakage of gadolinium was seen in these animals 
corresponding to the inflammatory changes around blood vessels. The moderate dose 
vector plus GCV animals exhibited much less impressive MRI alterations, and no cavity 
formation was seen by imaging or gross inspection corresponding to the lower toxic effect of 
the treatment regimen. These results indicate that MRI can be used to monitor tissue 
effects of this therapy. 
PCR Analysis of Tissues from Baboons Injected with Moderate and High Doses of 
ADV/RSV-tk 
Necropsy tissue (2-3 mm diameter) was used for the analysis. In cases of larger organ 
specimens (brain, liver, etc.) multiple (4 to 6) tissue samples were collected and pooled. 
Total DNA was isolated using SDS and proteinase K (Ausubel et al., 1987). A 1 ml aliquot 
of DNA from each sample was used in the PCR reaction. The primer oligonucleotides used 
were a sense primer Adv.3205 (5'GTGTTACTCATAGCGTAA3') and an antisense primer 
RSV 270A (5'GACTCCTAACCGCGACA3'), the former primer situated in the adenoviral and 
the latter in the RSV-LTR sequences and both 5' of the thymidine kinase gene in the 
recombinant plasmid pADL-1/RSV-tk used to generate the recombinant virus. The PCR 
reaction was carried out for 30 cycles of 30s @ 92°C, 30 s @ 50°C, and 1 m @ 72°C. The 
reaction mixture consisted of 50 mM of each dNTP nd 50 pM of each of the primers and 4 
units of Taq polymerase in a total volume of 100 ml (Saiki, 1990). At the end of PCR a 10ml 
aliquot of the product was electrophoresed on a 4% NuSieve agarose gel. The gel was 
stained with ethydium bromide and visualized under UV light. Those samples that yielded a 
232 bp fragment as seen on the gel were scored positive. The site of injection was positive 
in 3 of the 4 animals that received high doses of virus. One animal that had virus sequence 
at the injection site also had virus in its spinal cord. The other two animals that were positive 
at the injection site were negative in other areas of the CNS. No other tissues, including 
gonadal tissue, were positive for ADV/RSV-tk sequences. The results of the analysis are 
listed in Table 1. 
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Recombinant DNA Research, Volume 20 
