Project description:We generated a new mouse model with three copies of the HA epitope and eGFP knocked-in frame into the endogenous mouse Pkd1 gene by CRISPR/Cas9. The modified allele is fully functional. Using nanobody-coupled beads and large quantities of tissue (8 heads of P1 newborn mice / experiment / condition), we immunoprecipitated a PC1 interactome that was characterized by mass spectrometry.
Project description:Identifying Smad4-binding proteins in mouse CD4+ T cells under IL6SB condition. Mouse CD4+ T cells were isolated from mouse spleen and lymph nodes.
Project description:We developed a method to identify chromatin-associated proteins by mass spectrometry. In addition to many known DNA and chromatin-binders, we identified several RNA-binding proteins in the chromatin composition of mouse embryonic stem cells (mESC). Interestingly, we found Dazl as a RBP that specifically binds to chromatin in mESC grown in 2iL condition. So far, Dazl was reported as a RBP that regulates the stability of transcripts in cytoplasm. To identify the binding sites of Dazl on the genome, we carried out a ChIP-seq experiment. As a result, we detected about 1300 reproducible Dazl ChIP-seq peaks. Interestingly, most of the peaks are located close to the transcription start sites of developmental and pluripotency-related genes.
Project description:Autosomal recessive polycystic kidney disease is a severe, monogenetically inherited kidney and liver disease and PCK rats carrying the orthologous mutant gene serve as a model of human disease. We combined selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis of imaging data sets for identification of candidate lipid markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. Beside increased levels of serum-cholesterol these sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases suggesting that they be molecular markers of the disease. Genome-scale gene expression profiling of PCK and SD livers as control was performed to attempt elucidation of some of the underlying mechanisms leading to increases of cholesterol and taurine-conjugated bile acids in the PCK rat. Several pathways were found to be changed in cystic livers with up regulation or down regulation of important gene sets. Enhanced expression of steroid biosynthesis genes might result in the observed increased levels of cholesterol. In contrast, primary bile acid biosynthesis was found to be down regulated in diseased livers. These findings might be explained by compensatory mechanisms of liver metabolism to reduce toxic levels of accumulated bile acids. Snap-frozen liver tissue of 10 week old rats were subjected for RNA extraction and hybridization on Affymetrix microarrays to perform genome-scale gene expression profiling of n = 6 diseased PCK and n = 6 Sprague Dawley rat livers as control.