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Titolo:
SPONTANEOUS SILENCING OF HUMANIZED GREEN FLUORESCENT PROTEIN (HGFP) GENE-EXPRESSION FROM A RETROVIRAL VECTOR BY DNA METHYLATION
Autore:
GRAM GJ; NIELSEN SD; HANSEN JES;
Indirizzi:
HS HVIDOVRE HOSP,DEPT INFECT DIS,KETTEGAARD ALLE 30 DK-2650 HVIDOVRE DENMARK HS HVIDOVRE HOSP,DEPT INFECT DIS DK-2650 HVIDOVRE DENMARK
Titolo Testata:
Journal of hematotherapy
fascicolo: 4, volume: 7, anno: 1998,
pagine: 333 - 341
SICI:
1061-6128(1998)7:4<333:SSOHGF>2.0.ZU;2-M
Fonte:
ISI
Lingua:
ENG
Soggetto:
LEUKEMIA-VIRUS; CELLS; LYMPHOCYTES; INFECTION; GFP;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
26
Recensione:
Indirizzi per estratti:
Citazione:
G.J. Gram et al., "SPONTANEOUS SILENCING OF HUMANIZED GREEN FLUORESCENT PROTEIN (HGFP) GENE-EXPRESSION FROM A RETROVIRAL VECTOR BY DNA METHYLATION", Journal of hematotherapy, 7(4), 1998, pp. 333-341

Abstract

We have constructed a functional murine leukemia virus (MLV)-derived retroviral vector transducing two genes encoding the autofluorescent humanized green fluorescent protein (hGFP) and neomycin phosphotransferase (Neo), This was done to determine whether hGFP could function as amarker gene in a retroviral vector and to investigate the expression of genes in a retroviral vector. Surprisingly, clonal vector packagingcell lines showed variable levels of hGFP expression, and expression was detected in as few as 49% of the cells in a clonally derived culture. This indicated that hGFP expression was silenced in individual cells, This silencing could be diminished by selective culturing of the vector packaging cells with the neomycin analog G418 and was reduced bya 3-day treatment with the demethylating agent 5-azacytidine, The 5-azacytidine effect was transient, and hGFP expression in the vector packaging cells returned to untreated control levels within 2 weeks, Using flow cytometric analysis, hGFP expression was detected in up to 15% of transduced MT4 cells (a CD4+ lymphocytic cell line) after coculturing with packaging cells for 4 days. A 3-day postcoculture treatment with 5-azacytidine was shown to increase the hGFP-expressing MT4 cells from either 10.4% to 11.6% or 3.7% to 4.8%, corresponding to an increase in observed transduction efficiencies of 12% and 30%, respectively. These results indicate that silencing of gene expression from a retroviral vector may result from DNA methylation and occurs rapidly after transduction.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 02/12/20 alle ore 04:32:01