encoding the MoMLV structural genes ("gag/pol" and "env") present in the PCL, makes the 
probability of generating RCRV by homologous recombination extremely low. 
Multiple clones of cells transfected with gag/pol and env expression vectors were screened by 
protein immunoblotting (see Methods, 3.2). Individual cell clones were identified that expressed 
10-50x higher levels of gag/pol and similar levels of env compared with that of the PCL control 
PA317 (Fig 4). A number of these potential amphotropic or xenotropic PCLs were tested for their 
capacity to package retroviral vectors by measuring titer after transduction with the vectors KT-1 
and N2. After G418 selection, cell free supernatants were collected from the confluent monolayers 
and titered by measuring G418 r colonies formed after addition to either NIH3T3 TK- or HT1080 
cells. The cell clones with the highest titer were chosen as PCLs and referred to as DA (D17 
ampho) and DX (D17 xeno). 
In our hands, PA317 generated the highest titer of all the murine PCLs tested (Table IV, 
experiment 1). By direct comparison, the DA vector titers were as high as or higher than the titer 
produced from PA317 with both vectors tested (Table IV, experiment 2). In no case was RCRV 
detected from vector producing cells derived from DA. DA therefore has characteristics (high titer 
and undetectable RCRV) essential for human gene therapy trials. 
3.3. 1.2 Characterization of the human y-lFN vector producing cell line, 
VCLHuy. The y-IFN proteins are highly species-specific and therefore the human y-IFN gene 
must be used for human studies. The clonal vector producing cell line, VCLHuy, will be carefully 
characterized with regard to the possible presence of adventitious agents (summarized in Table I). 
The cell-free supernatant results in an average titer of 3.7x1 0 6 cfu/ml and several tests have failed 
to indicate the presence of RCRV. Co-cultivation of the VCLHuy vector producing cell line with 
Mus dunni cells (to amplify retrovirus, if present) also fails to detect RCRV. The cell line lacks 
adventitious agents as determined by broth and agar culture assays (for bacteria, fungi, and 
mycoplasma) as well as Hoechst staining (for mycoplasma). The cell line appears to have 1 
provector with the appropriate genetic structure (Fig 5). Vector from VCLHuy results in 
expression of biologically active y-IFN in transduced cells (Table III) with the appropriate genetic 
structure (Fig 5). The VCLHuy master cell bank will be characterized in accordance with the FDA 
"Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1987)". 
These and other tests are summarized in Table I. The high titer and lack of adventitious agents 
make VCLHuy ideal for the production of y-IFN vector for clinical trials. 
Recombinant DNA Research, Volume 17 
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