I want to visualize a protein’s localization and also identify the components of the complexes it forms at its endogenous expression levels in Drosophila. I would like to just swap out the stop codon of my GOI with a tag (Ab site, GFP, MS2 binding site), or a linker then a tag as to not interfere with protein function. Does anyone know if it is possible to do this with CRISPR in Drosophila? I want to avoid modifications to endogenous expression levels that result from third copy allele techniques.
Yes, it should be possible, we are planning a similar experiment. See the links below, can be done also in a tissue-specific manner.
http://www.nature.com/ncomms/2015/151217/ncomms10237/full/ncomms10237.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4728311/
Hi Bryan,
Yes, we've done the same for four different genes. Besides the very nice references above, I would include a recent one using the "classic" method; i.e.; adding homology arms to the donor plasmid:
Li Q, Barish S, Okuwa S, Volkan PC. Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging. G3 (Bethesda) 2015;5:2809–16. doi:10.1534/g3.115.021857.
Cheers
Antonio.
Thank you both, these papers were very helpful. I think I’ll probably go with the pT-STEP vector since I am also curious about the effects of miRNA sites in the 3’UTR so that vector will be perfect for those kinds of experiments.
I also found this paper very helpful for figuring out different ways to visualize the protein.
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