‘Knockout-first’ mouse model as a biological tool to study the role of KIAA0182 gene in hypoplastic left heart syndrome
von Fouzi Alnour
Datum der mündl. Prüfung:2016-03-16
Betreuer:Prof. Dr. Elisabeth Zeisberg
Gutachter:Prof. Dr. Steven Johnsen
Gutachter:Prof. Dr. Margarete Schӧn
EnglischObjective: Hypoplastic left heart syndrome (HLHS) is one of the most severe congenital heart diseases. The genetic etiology for this syndrome was strongly suggested by several previous studies, and a lot of genes were suspected to be involved in its pathogenesis. De novo mutation in KIAA0182 gene (Gse1 in mouse) was found in this disease, but its exact role in HLHS or any other biological process is still to be determined. The aim of this research is to investigate the consequences of KIAA0182 downregulation in vivo and in vitro. Methods: The ‘Knockout-first’ design was used in this study to produce supposedly null allele of Gse1 (Gse1tm1a(EUCOMM)Wtsi) in mouse model by disrupting its function on expression level. Human coronary arterial endothelial cells (HCAEC) were used to explore the role of KIAA0182 in endothelial to mesenchymal transition (EndMT), which was found previously to play a role in the development of endocardial fibroelastosis in HLHS. Ascending aorta constriction (AAC) was performed in mice as a model for cardiac fibrosis in vivo to confirm the results observed in vitro. Results: After several quality control tests to prove the correct targeting of Gse1 by the trapping cassette and to confirm its integrity, the genotyping results of 134 newborn mice from several matings between heterozygous (Gse1tm1a/WT) male and female using two different protocols indicated the presence of embryonic lethality in the homozygous mice (Gse1tm1a/tm1a). The expression level of Gse1 in the homozygous embryos was significantly downregulated compared with the wild type, but less than 50 % downregulation effect was observed, suggesting that the mutated allele Gse1tm1a behaved as a hypomorphic allele. That was associated with upregulation of Gse1 circular RNA, which might have the function of micro RNA (miRNA) sponge. KIAA0182 was activated in HCAEC upon treatment with TGF-β1 to induce EndMT, and KIAA0182 knockdown was associated with upregulation of some EndMT markers. However, no difference was found between heterozygous and wild type animals regarding fibrosis, systolic function or hypertrophy after AAC operation. Conclusions: The homozygousity of Gse1 trapping is associated with embryonic lethality, but further experiments are needed to prove the causal relation between Gse1 gene and this phenotype, where the altered expression of Gse1 circular RNA could also be involved. KIAA0182 gene may play a critical role in EndMT as a negative regulator, but confirming the importance of these findings in vivo requires further investigations.
Keywords: KIAA 0182; HLHS; Endocardial Fibroelastosis; EndMT; Circular RNA; ‘Knockout-First’ Mouse