In situ hybridization 
Marrow or peripheral mononuclear hematopoietic cells, after Ficoll-Bypaque 
separation, were adjusted to 2 x 10* cells/ml in Dulbecco's Minimal Essential 
Medium (Biofluids) containing 10% fetal calf serum (Gibco). Cells were 
transferred onto poly-L-lysine (Miles) coated slides by cytocentrifugaticn and 
fixed with freshly made 4% v/v paraformaldehyde (Sigma) in phosphate-buffered 
saline (Biofluids) containing 5 mM MgCl 2 (sigma) for 15 minutes at roan 
tenperature. The slides were stored at 4 # C in 70% v/v ETOH/H^. The methods 
of hybridization, washing, and exposure of film emulsion were those of Singer 
et al (24). The probe used was an **S-labelled neo R probe. 
T cell cloning 
T cell cloning was performed utilizing the methods previously described by 
Kernan et al (25) . 
Results 
Infection of Primate Hematopoietic Cells In Vitro 
Initial studies in our primate model were undertaken to establish whether 
and to what degree primate hematopoietic cells could be infected by the SAX 
p 
vector and express the vector-containing neo (neaiycin phosphotransferase, 
NFT) and h-ADA genes. Conditions were established for in vitro analysis of 
infection of hematopoietic progenitor cells, based on the resistance of cells 
p 
expressing the neo gene to the toxic neomycin analogue G418. As shown in 
Figure 2, the growth of normal cynomolgus marrow CFU-C (curve A) is completely 
suppressed at a G418 concentration of 0.29 mg /ml and above. 
Two protocols for infecting bone marrow progenitor cells in vitro were 
used. In the first, Ficoll/Hypaque-separated bone marrow mononuclear cells 
were cocultured for various periods of time with the SAX vector-producing NIH 
3T3 cell clone S3A. This producer cell line, derived from the PA12 araphotropic 
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Recombinant DNA Research, Volume 12 
