Linked Life Data

9403006

Source:http://linkedlifedata.com/resource/pubmed/id/9403006

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
10
pubmed:dateCreated
1998-1-5
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0145-2126
pubmed:author
pubmed:issnType
Print
pubmed:volume
21
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
951-9
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:9403006-Cloning, Molecular, pubmed-meshheading:9403006-DNA, Viral, pubmed-meshheading:9403006-Encephalomyocarditis virus, pubmed-meshheading:9403006-Flow Cytometry, pubmed-meshheading:9403006-Galactosides, pubmed-meshheading:9403006-Gene Transfer Techniques, pubmed-meshheading:9403006-Genetic Vectors, pubmed-meshheading:9403006-Humans, pubmed-meshheading:9403006-Immunophenotyping, pubmed-meshheading:9403006-Indoles, pubmed-meshheading:9403006-Lac Operon, pubmed-meshheading:9403006-Phenotype, pubmed-meshheading:9403006-Promoter Regions, Genetic, pubmed-meshheading:9403006-Protein Biosynthesis, pubmed-meshheading:9403006-RNA, Viral, pubmed-meshheading:9403006-Retroviridae, pubmed-meshheading:9403006-Simplexvirus, pubmed-meshheading:9403006-Thymidine Kinase, pubmed-meshheading:9403006-Transfection, pubmed-meshheading:9403006-Tumor Cells, Cultured, pubmed-meshheading:9403006-beta-Galactosidase
pubmed:year
1997
pubmed:articleTitle
Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector.
pubmed:affiliation
Department of Clinical Medicine, Pathology and Pharmacology, University of Perugia, Italy.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't