Etablierung und Charakterisierung eines Csrp2-defizienten Mäusestammes

The four cysteine rich proteins (CRPs), encoded by the CSRP-genes, form a subclass of the LIM-only proteins. These proteins mediate protein-protein interactions and are of fundamental importance for cell differentiation, cytoskeletal remodeling, and transcriptional regulation. This thesis examines the establishment and characterization of a Csrp2-deficient mouse strain that was created by insertion of a neomycin resistance gene cassette into exon 4 of the Csrp2 gene. The deficiency was proven on the DNA-, RNA- and protein-level. After backcrossing to generation N10 the respective mouse strain was transferred into the European Mouse Mutant Archive.It was shown that mice lacking a functional Csrp2 gene are viable and fertile. The frequency of homozygous nulls in the offspring of heterozygous matings is in accordance with the Mendelian genetics. A comparative analysis of the gene expression of the different Csrp genes was performed to verifiy the data from literature and to explore a potential related regulation of the other CRP-family members. The analysis revealed that Csrp1 was expressed in all organs tested, Csrp2 was found in kidney, liver, heart, lung, bladder, testis and ovar/uterus, and Csrp3 was expressed in heart and skeletal muscle, respectively. The good general condition of the Csrp2-ko. mouse might be caused by the adoption of the functions through CRP1 and/or CRP3. However, for both of them no change could be found in the amount of expressed mRNA or protein in the knock-outs.In skin fibroblasts and hepatic stellate cells, CRP2 has a dual subcellular expression pattern along the actin filaments and in the nucleus. In both cell types, no change of the structure of the actin-based cytoskeleton was induced as a consequence of the introduced deficiency. Since CRP-proteins are associated with important regulatory functions of cell differentiation, the hepatic stellate cells located in the liver have been examined more precisely. These cells specifically expressing Csrp2 play an important role in the development of liver fibrosis. In line with the transdifferentiation from HSC to MFB the increasing expression of Csrp2 could be demonstrated in cell culture. It was investigated via the CCl4 induced liver fibrosis model whether the knock-out of the Csrp2 gene had an influence on the susceptibility of fibrosis. The increased expression of Csrp2 in the treated wild type animals in comparison to the non-treated animals could clearly be shown. In all animals treated with CCl4, the fibrosis was well established. Via the analysis of appropriate marker genes no difference could be detected between wild type and knock-outs regarding the transdifferentiation in vitro and the susceptibility of fibrosis in vivo. Thus it can be assumed that the up-regulation of Csrp2 does not cause liver fibrosis since the Csrp2-nulls showed no altered tolerance towards liver fibrosis with the used model.Although the Csrp2-ko. mice showed no obvious, severe phenotype, there were small architectural changes detectable in the heart. Electronmicroscopic studies and alpha-actinin stainings in the heart showed that the Z-line is distinctive normal. Slightly enlarged cardiomyocytes and a marginal more folded intercalated disk with increased expression of the adherens junction proteins (beta-catenin and N-RAP) were found. Furthermore, enlarged heart fibers, slightly dilated thinner left ventricle and a diminished heart activity were detected. Together these changes in the heart demonstrate that the Csrp2-ko. mice develop a mild form of the dilated cardiomyopathy (DCM).In summary, it was shown that a functional CRP2 protein is not necessary for the development of the mouse embryo. Furthermore, it was demonstrated that the adult nulls display a mild cardiac phenotype. No alteration in susceptibility against toxins triggering hepatic fibrogenesis were observed.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Ludewig, Julia

Contributors dc:contributor

• Weiskirchen, Ralf

• info:eu-repo/classification/ddc/570
• Knockout <Molekulargenetik>
• Fibrose
• Biowissenschaften, Biologie

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-122886%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61208