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Titolo:
Long-term expression driven by herpes simplex virus type-1 amplicons may fail due to eventual degradation or extrusion of introduced transgenes
Autore:
Tsai, DJ; Ho, JJ; Ozawa, CR; Sapolsky, RM;
Indirizzi:
Stanford Univ, Dept Biol Sci, Stanford, CA 94305 USA Stanford Univ Stanford CA USA 94305 Dept Biol Sci, Stanford, CA 94305 USA
Titolo Testata:
EXPERIMENTAL NEUROLOGY
fascicolo: 1, volume: 165, anno: 2000,
pagine: 58 - 65
SICI:
0014-4886(200009)165:1<58:LEDBHS>2.0.ZU;2-5
Fonte:
ISI
Lingua:
ENG
Soggetto:
GENE-THERAPY; NEUROLOGICAL DISORDERS; RAT-BRAIN; IN-VIVO; VECTORS; ORGANIZATION; PROTECTS; NEURONS; SYSTEM; DNA;
Keywords:
gene therapy; herpes simplex virus; down-regulation; transgene; nuclear peripheralization; fluorescent in situ hybridization;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
25
Recensione:
Indirizzi per estratti:
Indirizzo: Sapolsky, RM Stanford Univ, Dept Biol Sci, Gilbert Bldg 428, Stanford, CA 94305 USA Stanford Univ Gilbert Bldg 428 Stanford CA USA 94305 4305 USA
Citazione:
D.J. Tsai et al., "Long-term expression driven by herpes simplex virus type-1 amplicons may fail due to eventual degradation or extrusion of introduced transgenes", EXP NEUROL, 165(1), 2000, pp. 58-65

Abstract

In gene therapy applications employing herpes simplex virus amplicon-basedvectors, a prevailing problem is the down-regulation of transgene expression over time. We have applied a combined immunocytochemistry and fluorescent in situ hybridization method to determine whether down-regulation of transgene expression at the single-cell level correlates with loss of vector DNA from the host cell nucleus. Utilizing separate fluorescent labels (i.e., rhodamine, fluorescein, and 4',6'-diamidino-2-phenlindole), we were able tosimultaneously detect transgenes, their products, and their locations relative to the nuclear compartment of a single cell. Detection of the reportergene lacZ and its encoded protein beta-galactosidase (beta-gal) was accomplished in in vivo experiments of the dentate gyrus of rats. A time course study of the expression of the transgene post-stereotactic microinfusion up to 60 days was made. Expression reached maximal levels within 12-24 h afterinfection, and lacZ presence was reduced to less than 3% of its maximal levels within 36 h after infection. In comparing days 1 and 60 post-stereotactic microinfusion, only one-fifth of the original DNA was observed in the area of a 100-mm radius around the site of microinfusion at day 60. Moreover, by comparing the locations of the reporter gene in cells that expressed the encoded protein versus those that did not, we found that introduced transgenes were preferentially localized in the nuclear periphery of down-regulated host cells, compared to expressing host cells. These results suggest that nuclear compartmentalization may play a role in the down-regulation of our reporter gene by means of peripheralization, extrusion, and/or degradation. (C) 2000 Academic Press.

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Documento generato il 21/01/20 alle ore 07:02:10