MAHansonHey #Drosophila Mastodon, I'm looking to make a Gal4 enhancer trap for Gal4 expressed under endogenous promoter. How would you go about this in 2024?
Ben de Bivort@MarkHanson Do you know the particular promoter already? Or do you want to screen enhancers?
@debivort specific promoter, specific gene target (DptB). I was thinking to use pScarless-HD-DsRed with double gRNA approach. That said, I can't seem to find an example plasmid with homology arms inserted just to double check my work. Also wondering if a more classic approach would be simpler.
dmz@MarkHanson @debivort There is already a CRISPR knock-in KozakGAL4 line for DptB available via @flystocks (BDSC #95042) from @kancaoguz and the Bellen lab. This is probably just as good as a T2A-GAL4, unless for some reason you need to make the GAL4 chromosome homozygous. If you still want to make a new reagent from scratch, you can check out the design of the targeting constructs in this paper: https://doi.org/10.1016/j.celrep.2019.12.018.
@dmz @debivort @flystocks @kancaoguz Holy crap this totally passed me by and is absolutely amazing! Thank you thank you thank you!!! Not just for this one construct, but some of the lines in the list of Gal4s they've generated will be incredibly useful for me down the line. WOW! 🤩
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@dmz @debivort @flystocks @kancaoguz
Quick Q based on the list I see: are you saying "unless you need to make it homozygous" because it's currently balanced? Or because these KozakGAL4 lines have known homozygous toxicity? I see some in the list aren't balanced...
In the case of DptB, I have existing mutants so for sure this isn't itself homozygous lethal/sterile. I'd happily spend the time to recombine it and clean for any off-target mutations/weirdness.
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@MarkHanson @debivort @flystocks @kancaoguz Because the GAL4 coding sequence replaces that of the endogenous gene in these (KozakGAL4) lines, any recessive phenotype associated with your gene of interest would presumably be uncovered in homozygous animals. So if you want to use a KozakGAL4 to study the expression pattern of your gene in a "wild-type" context, you would need to look at heterozygotes (as one usually does anyway when crossing to a UAS-effector line).
@dmz @debivort @flystocks @kancaoguz aye. In some cases due to off-target mutations in linkage, you can get homozygous toxicity. Or, in some cases, Gal4 homozygous is toxic, leading to eg melanotic timors (Lpp-Gal4)
But the lof to me is a feature, not a bug 🙂 planning to do UAS-based rescue with custom DptB transgenes where each has a polymorphism encoded etc... so homozygous (and so a stronger GAL4) is desirable.
Follow-up work to:
https://www.science.org/doi/10.1126/science.adg5725
Free link:
https://mahansonresearch.weebly.com/uploads/1/2/3/6/123603726/hanson_science.adg5725.pdf
@MarkHanson this just came out https://pubmed.ncbi.nlm.nih.gov/39180746/
Protocol for generation of CRISPR-Cas9-mediated specific genomic insertion of P2A-Gal4 to reveal endogenous gene expression in Drosophila - PubMed
Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the d …
@zandawala #Drosophila is an amazing community. "Hey, I'm thinking to do this and have the tools to, but wondering if there's a more direct way."
1) yes, and someone already did it for you in 2022
2) if you want here's a just-out paper going over that protocol and other options
😂