Add time:08/17/2019         Source:sciencedirect.com

In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal). We enginereed the U937 human cell line to co-express 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 μg/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES-LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level. Northern blot analysis of an X-Gal positive/ganciclovir sensitive 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 probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-β-d-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting. 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 transduced cells using a simple cytochemical stain.

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