RETROVIRAL TRANSFER OF HERPES-SIMPLEX VIRUS THYMIDINE KINASE AND BETA-GALACTOSIDASE GENES INTO U937 CELLS WITH BICISTRONIC VECTOR

In this study we describe a new retroviral vector utilizing an interna l ribosome entry site (IRES) from encephalomyocarditis virus to co-exp ress two genes. One is the herpes simplex virus type 1 thymidine kinas e gene (HSV-TK) which induces sensitivity to ganciclovir, and the seco nd is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with -bromo-4-chloro-3-indolyl-beta-D-gal actopyranoside (X-Gal), We enginereed the U937 human cell line to co-e xpress both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 mu g/ml) whi le X-Gal negative clones were not. Monoclonal cell lines showed a sing le copy of the provirus integrated in their genome with the TK-IRES-La cZ sequence stably inserted in all clones. The band distribution patte rn of the proviral DNA differed only at the long terminal repeat (LTR) level, Northern blot analysis of an X-Gal positive/ganciclovir sensit ive clone showed an mRNA band of 6 kb with both LacZ and TK probes, An X-Gal negative/ganciclovir resistant clone was negative with both pro bes. This report shows: (1) a therapeutic gene can be linked to a mark er gene by an IRES element achieving equivalent expression of both pro teins; (2) the co-expression of a marker gene makes fluorescein-di-bet a-D-galactopyranoside staining possible, and consequently separation o f cells expressing the LacZ gene by fluorescence activated cell sortin g, Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the tr ansduced cells using a simple cytochemical stain. (C) 1997 Elsevier Sc ience Ltd.