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 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-beta-D-galactopyranoside (X-Gal). We engineered 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 microgram/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-beta-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.
Di Ianni
M
M
Department of Clinical Medicine, Pathology and Pharmacology, University of Perugia, Italy.
Casciari
C
C
Ciurnelli
R
R
Fulvi
A
A
Bagnis
C
C
Sadelain
M
M
Lucheroni
F
F
Mannoni
P
P
Stella
C C
CC
Martelli
M F
MF
Tabilio
A
A
D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry; D004686:Encephalomyocarditis virus / Q000235:genetics*; D005434:Flow Cytometry; D005697:Galactosides / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D016130:Immunophenotyping; D007211:Indoles / Q000378:metabolism; D007763:Lac Operon / Q000235:genetics*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry; D012190:Retroviridae; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics*
D004279:DNA, Viral; D005697:Galactosides; D007211:Indoles; D012367:RNA, Viral; D013937:Thymidine Kinase; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside