TRANSFECTION AND HEAT-INDUCIBLE EXPRESSION OF MOLLUSCAN PROMOTER-LUCIFERASE REPORTER GENE CONSTRUCTS IN THE BIOMPHALARIA-GLABRATA EMBRYONICSNAIL CELL-LINE

Studies were initiated to begin developing a genetic transformation sy stem for cells derived from the freshwater gastropod, Biomphalaria gla brata, an intermediate host of the human blood fluke Schistosoma manso ni. 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'-fl anking region containing sequences indicative of a HSP promoter. Ident ified in the 5'-half (416 nucleotides) of this genomic fragment were T ATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-sh ock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron l ocated immediately 5' of the ATG start codon. To determine the functio nality of the putative snail promoter sequence, Bge HSP promoter/lucif erase (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 w ith 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 obtain ed only in the Bge HSP70 promoter/Luc-transfected cells following heat -shock. Results of transfection experiments demonstrate that the Bge H SP70(0.5k) DNA segment contains appropriate promoter sequences for dri ving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characteriz ation of an inducible promoter from a freshwater gastropod mollusc. Su ccessful 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.