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.
Highlights
Discoveries of gene function are most often driven by the integration of information related to cellular and subcellular protein expression patterns, genetic loss of function phenotypes, and protein–protein interactions
We present a detailed analysis of 200 tagged genes/proteins, and we performed a series of knockdown experiments on four tagged genes to assess the key features of this collection and demonstrate its power for functional analysis
We previously described MiMIC insertions in a coding intron of three genes that had recessive lethal phenotypes and showed that the lethality could be reverted by removing the gene trap cassette by RMCE (Venken et al, 2011a)
Summary
Discoveries of gene function are most often driven by the integration of information related to cellular and subcellular protein expression patterns, genetic loss of function phenotypes, and protein–protein interactions. Antibodies to individual proteins can be used to localize their expression and for protein interaction studies. Antibodies are not available for most proteins, and it is time consuming and expensive to generate them against a large number of individual proteins. An extensive survey reveals that antibodies are currently available against ∼450 different proteins in Drosophila, a mere ∼3% of all protein coding genes (Adams et al, 2000). An alternative to generating antibodies to individual proteins is to tag the genes with a common protein segment for which antibodies are commercially available.
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