Der kanalikuläre Gallensäurentransporter der Leber als Kandidatengen für den Gallensteinlocus Lith 1

Abstract

The formation of cholesterol gallstones is a polygenic disease. This implies, that the interaction of multiple genetic factors with the environment determines the quantitative expression of the phenotype. Gallstone genes co-localize with Quantitative Trait Loci (QTLs), chromosomal regions associated with cholesterol cholelithiasis. In this thesis a candidate gene for the cholesterol gallstone locus Lith 1, the gene encoding the canalicular bile salt export pump of the liver Abcb11, was characterized in detail and overexpressed in vivo by generating a BAC (bacterial artificial chromosome) transgenic mouse. In a molecular biological analysis, sequence information in unknown regions of the Abcb11 gene was generated and the genomic structure of the gene was determined by using DNA fragments cloned in plasmids or BACs as well as by adapter ligated PCR (genome walking). The sequences of the gallstone sensitive mouse strain C57L/J and the resistant strain AKR/J were compared by single strand conformation polymorphism (SSCP) analysis and by direct sequencing of PCR fragments. The cDNA of both strains is identical. In the 5' region of the gene a C/T polymorphism was detected at -3.757, and in intron 1 19 single nucleotide exchanges as well as a 5 bp insertion and a 3 bp deletion were identified. The functional relevance of these polymorphisms was investigated in dual luciferase assays by cotransfection with nuclear receptors and in electrophoretic mobility shift assays (EMSAs). The experimental procedures were complemented by in silico sequence analyses. Even though a regulatory potential was shown for the analyzed regions, their function for the differential transcriptional regulation between the inbred mouse strain could not be proven. In order to investigate the physiological effect of an increased Abcb11 expression on cholesterol homeostasis, a genomic BAC library was screened for Abcb11 positive clones by using a radioactively labelled probe. Subsequently, the BAC DNA was isolated and purified. After microinjection into pronuclei of selected gallstone resistant mouse strains, two Abcb11 BAC transgenic founder lines on the 129S1/SvImJ background were generated. Employing Southern and Northern hybridizations it could be demonstrated, that both lines have 1 - 2 extra gene copies apart from their endogenous Abcb11 copies, which are integrated into their genomes and expressed correctly. The transgenic mice show a normal development and no visible phenotype. By feeding a cholesterol rich lithogenic diet, cholesterol crystallization and gallstone formation were induced. Transgenic mice display a higher gallstone susceptibility in comparison to wildtype controls. The generation of the BAC transgenic mouse opens new perspectives for in vivo investigations. It will deliver an important contribution to the question of the identity of the cholesterol gallstone locus Lith 1 and the canalicular bile salt export pump Abcb11 as well as to the investigation of the complex protein interactions in biliary cholesterol homeostasis