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Titolo:
TRANSFECTION AND HEAT-INDUCIBLE EXPRESSION OF MOLLUSCAN PROMOTER-LUCIFERASE REPORTER GENE CONSTRUCTS IN THE BIOMPHALARIA-GLABRATA EMBRYONICSNAIL CELL-LINE
Autore:
YOSHINO TP; WU XJ; LIU HD;
Indirizzi:
UNIV WISCONSIN,SCH VET MED,DEPT PATHOBIOL SCI,2015 LINDEN DR W MADISON WI 53706
Titolo Testata:
The American journal of tropical medicine and hygiene
fascicolo: 3, volume: 59, anno: 1998,
pagine: 414 - 420
SICI:
0002-9637(1998)59:3<414:TAHEOM>2.0.ZU;2-#
Fonte:
ISI
Lingua:
ENG
Soggetto:
PSEUDOTYPED RETROVIRAL VECTORS; DOMINANT SELECTABLE MARKER; STABLE TRANSFORMATION; UPSTREAM PROMOTER; SHOCK GENES; HIGH-TITER; DNA; ACTIVATION; LIPOFECTION; EUKARYOTES;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
35
Recensione:
Indirizzi per estratti:
Citazione:
T.P. Yoshino et al., "TRANSFECTION AND HEAT-INDUCIBLE EXPRESSION OF MOLLUSCAN PROMOTER-LUCIFERASE REPORTER GENE CONSTRUCTS IN THE BIOMPHALARIA-GLABRATA EMBRYONICSNAIL CELL-LINE", The American journal of tropical medicine and hygiene, 59(3), 1998, pp. 414-420

Abstract

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained fromthe B, glabrata embryonic (Bge) cell line, we cloned from Bge cells acomplete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cellsusing a homologous, inducible promoter sequence now paves the way fordevelopment of methods for stable integration and expression of snailgenes of interest into the Bge cell line.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 20/09/20 alle ore 20:19:03