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Dept of Molecular Pharmacology
Dept of Microbiology & Immunology

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8. Improvements to retroviral producer lines:
Phoenix Systems.

First, 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 potential.

Gag-pol was introduced with hygromycin as the co-selectable marker and the envelope proteins were introduced with diphtheria resistance as the co-selectable marker.



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