Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/41521
Wiedemann-Steiner syndrome (WSS) is a Mendelian disorder of the epigenetic machinery. WSS is caused by heterozygous de novo mutations in the KMT2A gene which encodes a histone-methyltransferase 2A. WSS patients have characteristic facial features, growth retardation, intellectual disability, and hypertrichosis amongst other medical features. Other Mendelian disorders of the epigenetic machinery (Kabuki syndrome, Rett syndrome and Rubinstein-Taybi) have previously been reported to demonstrate a postnatal rescue of the neurological phenotype in mouse models. However, this has never been tested for WSS which demonstrates a considerable phenotypic overlap with these diseases and the question remains whether WSS is potentially another treatable cause of intellectual disability.
In collaboration with Jackson laboratories, we designed and created a novel mouse model of WSS (Kmt2a+/-LSL) using CRISPR-Cas9. The model contains a cassette with a terminating sequence surrounded by loxP sites, inserted into intron 1 of Kmt2a. This cassette insertion hinders the correct mRNA expression levels, mimicking a loss-of-function variant in Kmt2a, as seen in WSS patients. The mice are bred with an inducible Cre mouse model, which has the Cre recombinase fused to a modified estrogen receptor, that will transport to the nucleus and cut at the loxP sites once the mice are exposed to tamoxifen. The cassette can therefore be postnatally removed to regain KMT2A mRNA expression and potentially rescue previously observed phenotypes in the mice. Four optimizations were conducted in regard to the amount of tamoxifen given, the incubation time and injection route to the mice. Methods to detect the recombination of the cassette were developed, observing KMT2A expression at mRNA and protein levels, as well as the quantity of the cassette in the gDNA of the mice.
The cassette was sequenced to confirm correct insertion as well as off-target sites to confirm any results do not stem from off-target effects. The mouse model bread well and the stress of the injections did not affect the overall weight of the mice. Furthermore, the results demonstrate that loxP-Stop-loxP cassette can be postnatally removed, which can lead to rescued KMT2A mRNA levels. After reviewing many methods, the best way to evaluate KMT2A expression involves qPCR on mRNA from mouse samples while qPCR on gDNA proved an inconsistent method and was disregarded. With this thesis, the functionality of the cassette has been confirmed however, this model will require further optimizing to reach the full potential of KMT2A rescue.
|Masters_thesis_final_Skemman.pdf||2.66 MB||Lokaður til...31.05.2027||Heildartexti|