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Prikvačeni tweet
My PhD started with a bit of a whim. Marko Brankatschk
@Mekki1909 had just gifted us his collection of flies with endogenously YFP-tagged Rabs and we figured - what if you used a GFP-RNAi to knock down Rabs indirectly via the tag? Should work ... right?#AcademicTwitterPrikaži ovu nitHvala. Twitter će to iskoristiti za poboljšanje vaše vremenske crte. PoništiPoništi -
I would also take a copy
https://twitter.com/DresdenNGM/status/1222410343978356736 …Hvala. Twitter će to iskoristiti za poboljšanje vaše vremenske crte. PoništiPoništi -
An follow-up to this: Now that the article has its DOI, the supplemental data can be accessed on Figshare. https://doi.org/10.25387/g3.11339765.v1 … Includes raw phenotype counts, sequencing primers and the two supp. figures (although those are also in the Early Online PDF).https://twitter.com/benediktbest/status/1221397804687511552 …
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Of course, feedback of any kind is welcome. Has your lab thought about going with an indirect knockdown approach before? Now that endogenously GFP-tagged variants are more common, it starts being a viable alternative to mutants or deletions with less worry about false positives.
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Idk what happened with the image here. Twitter seems to pretend you can move the thumbnail but apparently it just deletes the pic altogether.pic.twitter.com/u6EYCXmf4H
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And that's two years of a PhD finally published. The most beautiful thing is that due to the indirect setup, our confidence in all observed effects being true positives is very high. There's probably false negatives, nothing is perfect. BUT IT FEELS GOODhttps://doi.org/10.1534/g3.119.400967 …
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But it still seems to disable the protein. At least when targeting YFP-Rab19, RNAi and nanobody produced knockdown phenotypes with the same severity. And combining both did similar to RNAi alone. (kudos to
@EstimationStats for the nice visualisation of effect size)pic.twitter.com/r6ceDALodq
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RNAi isn't the only way to knock things down though. Since we were targeting YFP, we figured it's worth comparing how well a GFP-RNAi does compared to a degrading GFP-nanobody. Or both combined. Tested on YFP-Rab1, the nanobody didn't actually eliminate the YFP fluorescence...pic.twitter.com/jJXRASfcWN
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So where are we at? Yes, indirectly knocking things down via an endogenous tag works. You might discover limitations of your tools that you weren't aware of before. Also, it makes finding maternal effects relatively easy (mixing tagged and untagged alleles in mothers).
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btl is expressed strongly during embryonic strages, then expression goes down during larval stages. We didn't test this, but I think probably, like btl, btl-Gal4 expresses less in larvae - hence lower RNAi efficiency, and lower impact while terminal cells grow.
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A big difference between these defects and terminal cell morphology is that the dorsal branches grow during embryogenesis (and determine number and position of terminal cells), while terminal cells grow their branches during larval stages. We used the btl-Gal4 driver.
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All of these abnormalities showed up in wildtype larvae sometimes. They're part of normal morphological variability. But a lot of Rab knockdowns increased the frequency (table shows mean frequency per larva, black frames are significant increases compared to negative control).pic.twitter.com/rosdbdoQpX
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Normally two opposing dorsal branches meet and fuse. That often failed. Or they fused not with the opposing but with the neighbouring branch. Besides that, excess terminal cells or missing ones - or misplaced ones.pic.twitter.com/CPGvOp1Cd7
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So we went through again, and recorded everything relating to the number and position of terminal cells. At this point I need to mention that we only looked at a subset of terminal cells, the ones located on the dorsal tracheal branches. And there was more than just excess cells.
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Cool, but we were expecting more. What about those extremely important Rabs? Like Rab1, Rab4, Rab7? Endosome, Golgi and ER organisers and such. No phenotypes whatsoever. Well, not in terminal cells. We noticed there were a bit... too many of them in Rab7 knockdowns.
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Out of 27 tested Rabs, four were hits. Rab8 and Rab6 had pretty nasty and interesting knockdown phenotypes. Rab2 was like a weak Rab6 knockdown, Rab10 like a really weak Rab8 knockdown.pic.twitter.com/9UuGFmRwrQ
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So first of all this turned out a tiny bit more work than expected. Like most PhD projects.
#phdlife But I got it done, and it worked. Just not like we expected. In short:Prikaži ovu nitHvala. Twitter će to iskoristiti za poboljšanje vaše vremenske crte. PoništiPoništi -
So they're: - epithelial type (tube is apical, outside is basal) - huge - a bit like neurons but with twice the membrane and less zapping Must be a somehow specialised membrane delivery system to build and maintain that, no? Rabs deliver membrane. Sounds good to go.
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It actually started sounding like a better idea every time we thought about it. My subject was the terminal cells of the larval fruit fly tracheal system. They form extremely long branches with air-filled tubes inside - the functional equivalent of blood capillaries.pic.twitter.com/T4IhI12C1Q
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