(single restriction site in LNL6) . The 1.6 kb 
Hindlll-BamHI fragment of pNeo (P-L Biochemicals) 
will be used as a probe. 
3T3 Amplification The use of 3T3 amplification in 
detecting replication-competent retrovirus in 
primates has been previously reported (see Appendix 
5 and 6) . In brief, 3T3 cells are maintained in 
Dulbecco's Modified Eagle's Medium with 10% fetal 
calf serum and 2 mM L-glutamine and grown at 37 °C, 
in 5% C0 2 . 3T3 cells are plated at 2xl0 5 cells in 60 
mm dishes twenty four hours prior to sample 
exposure. Bone marrow cells are tested for virus by 
cocultivating with the plated 3T3 cells overnight 
in a volume of 2 ml with polybrene (8 ug/ml) . After 
incubation the bone marrow cells are removed and 4 
ml of fresh medium is added. 3T3 cells are split 
1:10 when confluent and carried for at least 1 
weeks. When plates are confluent, fresh medium is 
added to the well and collected 20 hours later for 
analysis in the S+/L- assay (see Appendix 5) . 
Western Blot Analysis Evidence of infection with 
replication-competent murine amphotropic retrovirus 
will be performed using p30 antigen purified from 
amphotropic viral supernatant as previously 
described (see Appendix 5 and 6) . The antigen 
preparation is separated by 12% SDS-PAGE, 
transferred to nitrocellulose (70 min, 250 mAm, 60 
V in 0.25 M Tris, 0.22 M glycine, 0.0015% SDS, 10% 
methanol) . After transfer the filter is incubated 
for 1 hour with BLOTTO. To determine if antibodies 
to the viral p3 0 antigen is present, the 
nitrocellulose membrane is incubated with human 
serum diluted 1:5 in BLOTTO and subsequently 
incubated with anti-human IgG conjugated with 
peroxidase (Vectastain) . 
Methvlcellulose Assay Untreated and LNL-6 treated 
bone marrow and/or leukemic cells will be cultured 
in methylcellulose as previously described 
(Appendix 7) except for the addition of GM-CSF (100 
units/ml) , IL-3 (100 units/ml) , human 
erythropoietin (1 unit/ml) substituting for 
lymphocyte conditioned media. Leukemic cell density 
will vary between 10 4 to 10 5 cell/ml (to allow for 
variability in plating efficiency) and the 
susceptibility to G418 will be assayed at two 
concentrations (800 ug/ml and 1,200 ug/ml). 
Recombinant DNA Research, Volume 15 
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