Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector

Mauro Di Ianni, Carla Casciari, Raffaella Ciurnelli, Antonella Fulvi, Claude Bagnis, Michael Sadelain, Francesca Lucheroni, Patrice Mannoni, Carmelo Carlo Stella, Massimo F. Martelli, Antonio Tabilio

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)951-959
Number of pages9
JournalLeukemia Research
Volume21
Issue number10
DOIs
Publication statusPublished - Oct 1997

Keywords

  • Bacterial beta-galactosidase gene
  • Bicistronic vector
  • Herpes simplex virus-thymidine kinase gene
  • IRES sequence
  • Retroviral transfer
  • U937

ASJC Scopus subject areas

  • Cancer Research
  • Hematology
  • Oncology

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