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 |
