2. 3. 1.3 Packaging 
Because recombination events may lead to the production of a 
replication-competent virus, a safe and efficient amphotropic 
packaging cell line is necessary for transfer of exogenous genes 
into human cancer cells. We used a packaging cell line 
constructed so the gag-pol and env genes are separated on two 
different plasmids 28 . For this protocol retroviral supernates from 
the PA317 packaging cell line will be used. This packaging cell 
line has received prior approval for use in human gene therapy 
clinical trials. The presence of functioning retroviral genes in the 
packaging cell line will be monitored by an assay for reverse 
transcriptase production and by immunoprecipitation of env 
protein by metabolic labeling and immunoprecipitation with anti- 
env antiserum 28 . Continued absence of infectious virus will be 
determined from transfection-infection experiments. A neo- 
containing vector will be transfected into PA317 cells; colonies 
will be selected with G418. The supernate will be used to infect 
NIH 3T3 cells. Selection with G418 will be done after one month 
to ensure the survival of rare recombinants that do not have the 
neo gene but subsequently infect neo - positive cells. Supernate 
from the infected NIH 3T3 cells should not be infectious. These 
secondary supernates will be used to infect naive NIH 3T3 cells. 
Lack of infectivity will indicate absence of replication competent 
virus. Previous human studies have used the PA317 producer 
cell line. We will convert to this cell because of the extensive 
experience with its use and prior approval for human use. 
Supernate from the packaging cells will be produced in serum- 
free medium. Clones will be established and lots of frozen 
supernate prepared. 
2.3. 1.4 Preclinical studies 
The 2 Kb K-ras fragment (genomic exons 2 and 3) with a /?-actin 
promoter was cloned into the LNSX retroviral vectors in both 
orientations. The p53 cDNA with its /?-actin promoter was 
cloned into the LNSX retroviral vectors in both orientations. The 
LNSX-AS-K -ras was successfully packaged in the GP + envAm12 
packaging cell line. Initial titers ranged up to 10 7 . The 
constructs were then transduced into H460a cells. Specific 
expression of K-ras AS RNA was shown by slot blot analysis 
using vector only negative controls and a /?-actin probe for a 
loading control. Western blotting studies showed that 
expression of the K-ras p21 protein was specifically reduced. 
Next the effect of multiple cycles of transduction on transduction 
efficiency was assessed. Transduction efficiency was assessed 
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Recombinant DNA Research, Volume 16 
