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


Authors: Di Ianni, M.; Casciari, C.; Ciurnelli, R.; Fulvi, A.; Bagnis, C.; Sadelain, M.; Lucheroni, F.; Mannoni, P.; Stella, C. C.; Martelli, M. F.; Tabilio, A.
Article Title: Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector
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.
Keywords: human cell; nonhuman; flow cytometry; animal cell; phenotype; tumor cells, cultured; transfection; genetic vectors; cloning, molecular; beta galactosidase; histochemistry; simplexvirus; protein biosynthesis; immunophenotyping; dna, viral; thymidine kinase; indoles; herpes simplex virus; ganciclovir; rna, viral; retrovirus; retroviridae; leukemia cell line; fluorescein; virus vector; gene transfer techniques; lac operon; galactosides; beta-galactosidase; ribosome; promoter regions (genetics); fluorescence activated cell sorter; humans; human; priority journal; article; pyranoside; provirus; encephalomyocarditis virus; retroviral transfer; bacterial beta-galactosidase gene; bicistronic vector; herpes simplex virus-thymidine kinase gene; ires sequence; u937
Journal Title: Leukemia Research
Volume: 21
Issue: 10
ISSN: 0145-2126
Publisher: Elsevier Ltd  
Date Published: 1997-10-01
Start Page: 951
End Page: 959
Language: English
DOI: 10.1016/s0145-2126(97)00074-x
PUBMED: 9403006
PROVIDER: scopus
DOI/URL:
Notes: Article -- Export Date: 17 March 2017 -- Source: Scopus
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  1. Michel W J Sadelain
    538 Sadelain