SIFT analysis of NOTCH3

STSM Photo

Julie Rutten, Netherlands

2nd year PhD student, LUMC, Leiden

From the 14th of July until the 20th of September 2013, I have worked at the Institute for Stroke and Dementia research (ISD, Ludwig – Maximilians – University ) in Munich, using funding from the short term scientific mission (STSM ) program from COST. My project focuses on the development of antisense – mediated exon skipping as a potential therapeutic approach for CADASIL, a hereditary dementia and stroke syndrome caused by mutations in NOTCH3 . The goal of our exon skipping strategy is to prevent or reduce toxic NOTCH3 accumulation, which is believed to be the major problem underlying CADASIL pathology. Together with the CADASIL research group in Munich, we have set up a collaboration to further characterize the modified NOTCH3 protein after exon 4-5 skipping. Specifically, we want to test our hypothesis that exon 4-5 skipping leads to a modified protein that does not aggregate.

The aim of my 10 week visit to the ISD, was to prepare and set up aggregation analysis of NOTCH3 constructs, using SIFT (scanning for intensely fluorescent targets). For this purpose, we generated constructs of small fragments of NOTCH3 lacking exons 4 and 5. We found that these NOTCH3 fragments are expressed by HEK cells and are secreted into the cell culture medium, the latter being a prerequisite for SIFT analysis. The constructs have been purified and subsequently labeled with a dye for protein detection and aggrega tion analysis in the SIFT reader. In addition, to analyze translation of the modified protein, we cloned constructs of full length NOTCH3 lacking exons 4 and 5. We found that the modified NOTCH3 protein is expressed after transient transfection in HEK 293 cells .

Personally, this STSM has been an inspiring and very constructive experience which I can recommend to any other young researcher. Regarding our exon skipping project, both the small and full length NOTCH3 constructs that were made, are invaluable for further analysis of the modified NOTCH3 protein after exon skipping. Finally, a fruitful and continuing collaboration with ISD has been set up, also thanks to the STSM project.

July 2013