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Elife. 2017 May 31;6. pii: e26420. doi: 10.7554/eLife.26420.

A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells.

Author information

1
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
2
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
3
Department of Neuroscience, Baylor College of Medicine, Houston, United States.
4
Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
5
Jan and Dan Duncan Neurological Research Institute, Texas Children's Hospital, Houston, United States.

Abstract

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila. Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

KEYWORDS:

D. melanogaster; FLEx; MiMIC; SNF4Aγ; Trim9; developmental biology; effete; neuroscience; post-mitotic cells; stem cells

PMID:
28561736
PMCID:
PMC5493436
DOI:
10.7554/eLife.26420
[Indexed for MEDLINE]
Free PMC Article

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