no longer detectable after day 160 in either animal. NFT activity was also 
detected early in the post-transplant period (day 52) but was not detected at 
day 104 at a time when h-ADA was still discernible (Figure 7) . 
In an attempt to determine the frequency of cells expressing the vector 
encoded RNA, situ hybridization was performed using a -^^S-labelled-neo^ 
probe on PBMC obtained from animal #57 on day 127 (Figure 8) . Hybridization 
was detected in 28 of the 3415 (0.8%) cells counted. 
To define the lineage (s) of cells containing the vector, we examined 
marrow CFU-C and clonable peripheral blood T cells for evidence of G418 
resistance. Assays of marrow from both animals #57 and #58 for G418 resistant 
CFU-C performed on days 28, 104 and 169 failed to demonstrate myeloid colonies 
resistant to the same high concentration (1.7 mg/ml) of G418 that was used to 
detect CFU-C cultured from vector-infected marrow at the time of 
transplantation (Figure 2, Table 2). However, with concentrations of G418 
lower than maximal (0.29 mg/ml), but still toxic for non-inf ected controls, 
resistant colonies were obtained frcm monkey #56 at day 28 and 169 post 
transplant, and frcm monkey #57 at day 169 post transplant (Table 2) . 
In limiting dilution analyses of peripheral blood lymphocytes as a means - 
of quantitating the frequency of clonable T lymphocytes expandable in the 
presence of mitogen (PHA) and IL-2, clonable T cells resistant to 
concentrations of 0.1 mg/ml G418 were detected in animal #56 at a frequency of 
1/93 on day 181 post-transplant (Figure 9) • By comparison, the overall 
frequency of clonable T cells detected without selection in the PBMC from this 
animal was 1/7. Thus, approximately 8% of the clonable T cells detected in the 
circulation were resistant to 0.1 mg/ml G418. In contrast, assays of normal 
cyncmolgus monkey lymphocytes have consistently failed to detect clonahle T 
cells resistant to this concentration (0.1 mg/ml) of G418. Further analysis 
roftfll Recombinant DNA Research, Volume 12 
