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Elife. 2015 Mar 31;4. doi: 10.7554/eLife.05338.

A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Author information

1
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.
2
Program in Developmental Biology, Baylor College of Medicine, Houston, United States.
3
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.
4
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
5
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, United States.
6
Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States.

Abstract

Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

KEYWORDS:

D. melanogaster; RMCE; bruchpilot; cell biology; deGradFP; discs large 1; dunce; iGFPi; neuroscience

PMID:
25824290
PMCID:
PMC4379497
DOI:
10.7554/eLife.05338
[Indexed for MEDLINE]
Free PMC Article

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