murine amphotropic (MA, derived from the cloned hybrid MoMLV-4070A provirua, pAM) (50) 
retrovirus controls. 
3. 2. 2. 4. ASSAY FOR RCRV IN THE VECTOR-PRODUCING CELL LINE, VCLHUy. Since 
amphotropic retroviruses would be unable to replicate efficiently on an amphotropic PCL (57), it 
was possible that very low levels of RCRV existed below the detection limits. To test this we co- 
cultivated 5x10 s VCLHuy vector producing cells with an equal number of Mus dunni cells that are 
known to efficiently support the replication of all classes of murine RCRVs (60). Every 3-4 days 
the cell number was split to 5x10 s . After 10-14 days of this "amplification", the supernatants were 
removed and tested for RCRV by either the standard S+L- or the MdH assay, described above. 
3.2.3. Protein Expression Assays 
3.2.3. 1. MURINE CLASS 1 MHC Expression ASSAYS. RIPA cell extracts were separated on 
10% acrylamide gels and blotted onto Immobilon transfer membranes essentially as previously 
described (61). Detection of Class I MHC proteins in mouse cells was carried out by incubation 
with a rat monoclonal antibody 7.2.14 (62) and 125 I protein A. The epitope detected by the 
antibody was encoded within a region that is well conserved between murine MHC haplotypes. 
Alternatively, cells were stained with the 34.4 anti-D d antibody (63) and binding measured by 
FACS analysis with fluorescene isothiocyanate conjugated rabbit anti mouse IgG (Capell). 
3. 2. 3. 2. BIOLOGICAL ACTIVITY OFy-IFN. y-IFN activity was quantified by measurement of the 
protective effect against cytotoxic infection with encephalomyocarditis virus (64). 
3.3 RESULTS AND DISCUSSION 
3.3.1 Retroviral Vector Producing Cell Lines 
3.3. 1.1 PACKAGING Cell Lines. Due to the safety risks associated with generating 
amphotropic or xenotropic RCRV by homologous recombination with endogenous sequences, we 
have developed MLV-based retroviral PCLs from a canine cell line, D17. There are no known 
canine retroviruses and at least one canine fibroblast cell line Cf2Th, ATCC CRL 1430, has been 
shown by us to lack genomic sequences homologous with MLV by high stringency genomic 
hybridization analysis (data not shown). To further decrease the chance of generating RCRV we 
have generated PCLs which contain genetically unlinked MLV structural genes ("split genome") 
that have minimal sequence homology with the vector DNA. The resultant requirement for 
multiple homologous recombinations between the therapeutic pro-vector DNA and the DNA 
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Recombinant DNA Research, Volume 17 
