3.2. 1.3 Generation of Retroviral Packaging Cell lines. 
MLV structural gene expression vectors. To minimize the possibility of forming RCRV resulting 
from genetic interactions between the MLV pro-vector DNA and the structural genes of the PCL, 
separate expression vectors, each lacking the viral LTR, were generated to express the gag/pol 
(50), amphotropic envelope (51), or xenotropic envelope (52) genes independently. In order to 
express high levels of the MLV structural proteins in the host cells, the human CMV immediate 
early promoter was utilized (53). Transcription was terminated by the SV40 late termination signal 
(residues 2717-2363). A diagrammatic representation of these expression vectors is presented in 
Fig 3. 
Transfection of host cells for the generation of PCLs. A canine cell line, D17 (ATCC #: CCL 
183), was co-transfected with a 10X excess of the pSCVIO plasmid (Fig 3) and a methotrexate 
marker, pFR400 (54). After selection in HAT medium individual clones were isolated and tested 
for MoMLV gag expression by protein immunoblotting. A gag/pol positive clone was re- 
transfected with a 10X excess of either amphotropic or xenotropic envelope expression vectors 
(Fig 2) and a phleomycin resistance marker, pUT507 (55). Co-transfection of a 10X 
stoichiometric excess of the expression vector over that of a separate selectable marker was done to 
ensure high copy number of the expression vector in drug resistant cell clones. After selection for 
transfected drug resistant cells, individual drug resistant cell colonies were expanded and analyzed 
for MoMLV gag/pol expression by extracellular reverse transcriptase activity (modified from ref 
56), intracellular p30& a s by Western blot using anti p30 antibodies (goat antiserum #77S000087 
from the National Cancer Institute), and either intracellular gp80 env (amphotropic envelope) or 
gp75 env (xenotropic envelope) expression by Western blot using anti gp70 env (goat antiserum 
#79S000771 from N.C.I.). Independent clones that expressed high levels of gag/pol and env 
were transduced with the vector N2, and the clones with best titers that did not generate RCRV 
were identified. DA and DX cells are the MLV-based amphotropic and xenotropic retroviral PCLs, 
respectively, identified from these studies. 
3.2. l .4. Generation of vector producing Clones. 
Production of xenotropic pseudotvped vector by transient transfection. Highest titers are obtained 
when retroviral vectors are introduced into PCLs by transduction instead of transfection (50). 
Although amphotropic MLV vectors are known to infect the D 17 parent cell line, the DA PCL is 
blocked for infection by amphotropic vector as its receptor is blocked by the expression of 
amphotropic envelope by "viral interference" (57). To overcome this problem, vectors were used 
that enter the cell via a different receptor by utilizing another retroviral envelope (xenotropic 
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Recombinant DNA Research, Volume 17 
