Erzeugung von Maus-knock-out-Modellen für die Sulfatasen Sulf1, Sulf2 und Arylsulfatase G

Abstract

In this work, mouse knock out models for the sulfatases Sulf1, Sulf2 and arylsulfatase G (ASG) were generated by using the gene targeting method. Preliminary in vitro data have shown that Sulf1 plays an important role in embryonic tissue differentiation, homeostasis and other essential cellular processes, which rely on heparan sulfate dependent signal transduction pathways. Sulf2 shows extensive sequence homology to Sulf1, but its function is still unknown, as well as that of ASG.We have performed gene knock outs following the classical, non-conditional strategy. First, mouse cDNAs in form of EST-clones were characterised. Cosmids carrying sulfatase gene fragments were identified by screening of the 129/Ola mouse genomic cosmid-library with radioactively labeled cDNA-probes. The cosmids were checked for the presence of the relevant exon/intron regions by Southern blotting. Positive cosmids were characterised by restriction mapping and sequence analysis. 3.1 to 3.8 kb DNA fragments, carrying one of the two first exons of the sulfatase genes in central position, were subcloned and further used for construction of the gene-targeting vectors. The aminoglycoside phosphotransferase gene, rendering cells resistant to aminoglycoside antibiotics, such as G418, was inserted into the respective exon to disrupt the reading frame. These constructs were transfected into mouse embryonic stem cells by electroparation and the cells were cultured in G418-containing selection medium. Approximately 100 of G418-resitant ES clones (for each sulfatase) were isolated and screened for successful homologous recombination by Southern blotting with external as well as internal DNA probes. Positive ES cell clones were prepared for injections into blastocysts.After injection of the ES cells into blastocysts and retransfer to the uterus of pseudopregnant mice chimeric offspring was generated. These animals were interbred with C57 BL/6 mice to produce the heterozygous F1-generation. The chimeric mice were also interbred with 129/Ola mice to generate animals with pure 129/Ola genetic background. In the F2 generation animals homozygous for the SULF1 or SULF2 null allele were obtained with reduced frequency, differing Mendel's law predictions. Very recently, also the first ASG knock out mice were obtained and investigations of phenotypic effects will start soon. Sulf1 and Sulf2 knock out mice are fertile. SULF1-/- animals show no obvious phenotype, adult mice on average are 1.8g heavier than wild type mice of the same age. Approximately 40% of the SULF2-/- mice die at the age of 1-6 weeks and present various CNS abnormalities, such as hydrocephalus and hypo- or aplasia of Corpus callosum. The rest of SULF2-/- animals show no obvious phenotype. Double (SULF1-/- SULF2-/-) mutants could not be generated so far. Thus, deficiency of both sulfatases apparently is lethal.The generation of knock out mouse models for the Sulf1, Sulf2 and ASG sulfatases provides a basis for further investigations of the in vivo functions of these newly discovered enzymes. Derived cell lines are already being used for identifying the natural substrates of these sulfatases and for functional in vitro studies.