question regarding UAS-Gal4 system

[Carter Wheat]

Hello all,

I hope I've reached the right audience here with my question. I am a PhD student working in a structural biology lab, and have no experience working with drosophila. I am proposing a project working with drosophila proteins, and a leg of that project involves working with live flies. 

I am proposing on ordering several fly stock strains, in which some cause mild growth defects and some are lethal strains. I am hoping to introduce rescue proteins to these strains using the UAS-Gal4 expression system to observe their phenotypes. How would one go about introducing a gal4 line into potentially lethal fly strains? I am hoping to use a ubiquitous gal4 promoter. Are there any other major considerations to this process?

I hope that my question makes sense. Any help, or resources one could point me to would be greatly appreciated.

Thanks!

Carter

[Wan Hee Yoon]

Hi Carter,

We typically use ubiquitous Gal4 drivers, including da (daughterless)-Gal4, Actin-Gal4, and Tubulin-Gal4.
Tub-Gal4 and Act-Gal4 lead to very high levels of expression. In contrast, da-Gal4-mediated expression results in lower levels of expression than those from Tub- or Act-Gal4. Therefore, if your gene of interest has gain-of-function effects (phenotypes caused by overexpression of your wild-type gene), a weaker driver is a better choice.
I tested 3-4 different Gal4, including strong and weak drivers, to determine which drivers show the optimal rescue for loss-of-function mutants.

Double balancer flies can combine Gal4 or UAS-gene with your mutant alleles if located in different chromosomes.
If the mutant allele and UAS transgene or Gal4 drivers are located on the same chromosome, you need to recombine your UAS transgenes or Gal4 drivers with your mutant allele.

In addition, T2A-Gal4 mutants(Lee et al. eLife 2018; Diao et al. Cell rep. 2015; Yoon et al. 2017 Neuron) have worked well to rescue their loss by combining UAS-wildtype target genes.

Please let me know if you have more questions.

Best wishes,
Wan Hee