title: Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.
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 from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete 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 cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.
authors.affiliation: Department of Pathobiological Sciences, University of Wisconsin, Madison 53706, USA.
authors.forename: T P
authors.initials: TP
authors.lastname: Yoshino
authors.forename: X J
authors.initials: XJ
authors.lastname: Wu
authors.forename: H D
authors.initials: HD
authors.lastname: Liu
mesh_terms: D000818:Animals; D001483:Base Sequence; D001700:Biomphalaria / Q000196:embryology / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004199:Disease Vectors; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D008156:Luciferases / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D008156:Luciferases