we added the ability to monitor gag-pol production on a cell-by
cell basis by intro ducing an IRES-CD8 (32) surface marker expression
cassette downstream of the reading frame of the gag-pol construct.
IRES (internal ribosome entry site) sequences allow sec ondary
or tertiary protein translation from a single mRNA tran script
(33). Thus, CD8 expression is a direct reflection of intracellular
gag-pol and the stability of the producer cell populationÍs
ability to produce gag-pol can be readily monitored by flow
cytometry. Second, for both the gag-pol and envelope constructs
non-Moloney promoters were used to minimize recombination potential
with introduced retroviral constructs, and different promoters
for gag-pol and envelope were used to minimize their inter-recombination
was introduced with hygromycin as the co-selectable marker and
the envelope proteins were introduced with diphtheria resistance
as the co-selectable marker.