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Titolo:
The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts
Autore:
Dwarakanath, RS; Clark, CL; McElroy, AK; Spector, DH;
Indirizzi:
Univ Calif San Diego, Mol Biol Sect, La Jolla, CA 92093 USA Univ Calif SanDiego La Jolla CA USA 92093 l Sect, La Jolla, CA 92093 USA Univ Calif San Diego, Ctr Mol Genet, La Jolla, CA 92093 USA Univ Calif SanDiego La Jolla CA USA 92093 Genet, La Jolla, CA 92093 USA
Titolo Testata:
VIROLOGY
fascicolo: 2, volume: 284, anno: 2001,
pagine: 297 - 307
SICI:
0042-6822(20010605)284:2<297:TUORBF>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
IE2 86-KILODALTON PROTEIN; BACTERIAL ARTIFICIAL CHROMOSOME; BOX-BINDING-PROTEIN; EARLY GENE PROMOTER; TATA-BOX; TRANSCRIPTIONAL ACTIVATION; MAMMALIAN-CELLS; EFFICIENT GENERATION; REGULATORY PROTEIN; HERPESVIRUS GENOME;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
52
Recensione:
Indirizzi per estratti:
Indirizzo: Spector, DH Univ Calif San Diego, Mol Biol Sect, 0366,9500 Gilman Dr, La Jolla, CA 92093 USA Univ Calif San Diego 0366,9500 Gilman Dr La Jolla CA USA92093
Citazione:
R.S. Dwarakanath et al., "The use of recombinant baculoviruses for sustained expression of human cytomegalovirus immediate early proteins in fibroblasts", VIROLOGY, 284(2), 2001, pp. 297-307

Abstract

The isolation of viruses with mutations in essential genes requires that they be propagated in cells expressing the wild-type proteins. This has beena particularly challenging problem for studying mutations in the human cytomegalovirus (HCMV) immediate early (IE) gene, IE2 86. In the past, we tried a number of approaches to derive human fibroblasts expressing wild-type IE2 86, but were unable to maintain expression of a fully functional protein. To overcome this obstacle, we developed a strategy whereby recombinant baculoviruses were used as vectors for the expression of HCMV IE proteins in primary human fibroblasts (FFs). The IE2 86 and IEI 72 cDNAs, as well as the genomic fragment of the UL122-123 region under the control of a chicken actin promoter, were introduced into the baculovirus genome by site-specifictransposition in Escherichia coli. Recombinant "bacmid" DNAs were then transfected into Sf9 cells to generate recombinant baculoviruses. 558 infectedat high m.o.i, with these baculoviruses expressed high levels of the HCMV protein for at least 1 week, as determined by immunofluorescence assays andWestern blots. Moreover, the IE2 86 protein was found to be fully functional with respect to its ability to activate the HCMV UL112-113 early promoter. Recombinant baculoviruses expressing IEI 72 were also able to efficiently complement HCMV ie1 mutants. These data demonstrate the potential of using recombinant baculoviruses as vectors for the expression of toxic viral genes in human cells and for subsequent isolation of mutant HCMV lacking these essential genes, (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 18/09/20 alle ore 15:30:41