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Elife. 2018 Mar 22;7. pii: e35574. doi: 10.7554/eLife.35574.

A gene-specific T2A-GAL4 library for Drosophila.

Author information

1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
2
Department of Genetics, Harvard Medical School, Boston, United States.
3
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
4
Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States.
5
Howard Hughes Medical Institute, Harvard Medical School, Boston, United States.
6
Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
7
Department of Neuroscience, Baylor College of Medicine, Houston, United States.
8
Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States.

Abstract

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

KEYWORDS:

CRIMIC; D. melanogaster; GAL4/UAS; MiMIC; cassette excision; chromosomes; complementation; gene expression; loss of function

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