INCREASED PROBABILITY OF EXPRESSION FROM MODIFIED RETROVIRAL VECTORS IN EMBRYONAL STEM-CELLS AND EMBRYONAL CARCINOMA-CELLS

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (E S) cells. We compared levels of expression in PA317 fibroblasts, F9 (E C) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors base d on our previously reported MND backbone, which has alterations to ad dress three viral elements implicated as repressors of expression by M o-MuLV: the enhancer, the primer binding site, and the negative-contro l region. Expression was evaluated with three reporter genes, the chlo ramphenicol acetyltransferase (CAT) gene, whose expression was measure d by enzymatic assay and by Northern blotting; a truncated nerve growt h factor receptor (tNGFR), whose expression was measured by fluorescen ce-activated cell sorting (FAGS) as a cell surface protein; and the en hanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher leve ls of CAT activity (5- to 300-fold) and greater quantities of vector-s pecific transcripts in ES and EC cells transduced with the modified MN D-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT- SN are >80 times more abundant than the L-CAT-SN transcripts. FAGS ana lysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-t NGFR-SN, indicated that only 1.03% of the CCE cells containing the L-t NGFR-SN vector expressed the cell surface reporter, while the MND-tNGF R-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cel ls containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99. 89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Es sentially identical results were produced with an analogous pair of ve ctors encoding EGFP, In unselected pools of F9 cells 48 h posttransduc tion, the L-EGFP-SN vector drove expression in only 5% of the populati on while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion o f cells shelving expression from L-EGFP-SN decreased slightly to 3% wh ile expression from the MND-EGFP-SN vector persisted in 80% of the cel ls. Interestingly, in the few ES and EC cells which did show expressio n from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN ve ctor in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector M ND, achieves higher net levels of expression due to a greater frequenc y of expression, which may de useful for the expression of exogenous g enes in EC and ES cells.