Update: Measuring DNA repair capacity and visualizing huntingtin in HD patient cells

Blog post by Dr. Tamara Maiuri

Image credit: Justinas

Previously I described a method to measure DNA repair capacity in cells: the GFP reactivation assay. This worked nicely in mouse striatal cells, with HD cells consistently showing about half the repair capacity of wild type cells. I have since tried it in cells from HD patients, using a different method to measure the GFP signal (microscopy instead of flow cytometry). The results were similar: a lower repair capacity was seen in HD cells (see experiment on Zenodo). The difference wasn’t as big as with mouse striatal cells, which is to be expected from clinically relevant CAG lengths compared to a model system that exaggerates the effect of expanded huntingtin. But the experiment was done twice with very similar results each time. In the coming weeks I will test whether this small but consistent difference is exacerbated by treating cells with DNA damaging agents. I will also make sure we’re measuring DNA damage pathways, and not some other phenomenon, by knocking down or inhibiting PARP. Stay tuned…

I also previously reported a way to visualize huntingtin protein at sites of DNA damage: stable cell lines expressing an inducible, huntingtin-specific YFP-tagged intrabody. I’m happy to say that the stable cell lines are growing, albeit slowly. If the growth rates recover, we will have available TruHD-Q21Q18, TruHD-Q41Q17, TruHD-Q43Q17, and TruHD-Q50Q40 cell lines in which huntingtin protein can be visualized in real time by addition of doxycycline to the media. The slow growth may be because of the combined toxicity of nucleofection and G418 selection, or due to leaky expression of the intrabody, which interferes with cell division. I’m currently testing the first idea by lowering the G418 concentration. If this doesn’t work, I may have to use alternate methods of detecting endogenous huntingtin. Fingers crossed!


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