envelope). Xenotropic-pseudotyped retroviral vector (58) was generated by calcium phosphate 
transfection of DX cells with 5 ng of pHuy-IFN plasmid (described in 3.2. 1.1 above). After 2 
days, media containing xenotropic vector was collected off the cells and passed through a 0.45 pm 
filter. 
Transduction and selection of retroviral packaging cell line. For transduction, the cells were 
seeded at 1 x 10 5 cells/ 6 cm dish with 4 pg/ml polybrene. The following day, 0.2 ml of the 
supernatant collected off the DX transiently transfected cells (described in 3.2. 1.3. above) was used 
to transduce the Huy vector into the DA cells. The following day, the media was replaced with 
fresh media containing 800 pg/ml G418. Cells were grown to confluence and expanded under 
selection to 10 cm dishes. The cells were then cloned by limiting dilution, clones were screened, 
and a high titer, retrovirus-free producer clone was identified, VCLHuy. 
3.2.2. Vector Characterization 
3.2.2. 1. G418 RESISTANCE TITER ASSAY. Titer was determined by addition of serially diluted 
test samples onto lxlO 5 NIH3T3 or HT1080 cells seeded on a 6 cm diameter tissue culture dish. 
After 1 day the culture was placed in medium containing G418 and the resistant colonies were 
enumerated after 10-14 days without correction for cloning efficiency. Titers were routinely 
determined from at least 2 measurements, and were found to be essentially equivalent for 
amphotropic vector on the two cell types. 
3. 2.2.2. Characterization and Quality control (QC) testing. A working cell bank of 
VCLHuy will undergo complete characterization and safety evaluation testing, as described in 
Table I. Vector QC testing is described in Table II. Transduced autologous tumor cell 
characterization is described in Table III. 
3.2.2. 3. Assay for Replication-Competent Retrovirus (RCRV) in vector-containing 
SAMPLES. The presence of RCRV was tested in 3 ways: 1) by the standard S + L- assay using a 
mink cell line, MjCli (ATCC # CCL 64. 1), as described (59); or 2) a modification of the S+L- test 
in which test samples were first allowed to replicate (amplified) on the precursor cell line to MjCl^ 
Mv-l-Lu (ATCC # CCL 64), for 14 days before transferring medium to the test cell line, MjCli; 
or 3) by a marker rescue assay in which test samples were applied to a cell line, MdH, which was 
generated by delivering a Hyg r retroviral vector to Mus dunni cells (60). If RCRV is present, then 
the vector will be rescued from the cell line by trans-complementation, and cell-free supernatants 
will confer Hyg r to the target HT1080 cells. In our experience the sensitivities of all of these assays 
are comparable (1 viral unit per ml) and result in indistinguishable TCID 50 titers with positive 
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