Auxin-induced (AID) protein degradation in drosophila larvae
muriel.boube-trey, Denis Jullien, Adeline Payet, Emmanuelle Guillou, Henri-Marc Bourbon, Sandra Bernat-Fabre
Abstract
The present protocol describes how to operate the auxin degradation system in drosophila larvae.The auxin-induced degradation, also referred to as AID, is an efficient targeted protein degradation system widely used in many model organisms. Thanks to the fact that fast degradation is triggered once a small molecule, the auxin, is added to the cell environment, a tight temporal control of the loss of a protein of interest can be achieved. This unique control of when the proteolysis is triggered allows to study the precocious consequences of the loss of function. Importantly, the implementation of this protocol requires genetically modified flies that express the auxin-dependent F-box protein TIR1, usually under the control of the UAS/Gal4 system to achieve a spatial control of the degradation, and in which the AID degron has been inserted (typically using CRISPR) to the coding sequence of the gene of interest. The problem raised by the use of the AID system in drosophila larvae, as in all the metazoans that have thick tegument, is the penetration of the auxin to each indidual cell of the organism. Here we present a non-invasive strategy based on the ingestion by the larva of a nutritive medium that contains auxin. We detail how to prepare the auxin containing food, handle the larvae, and which food container to use. Our method allows a fast degradation in the imaginal discs, as early as 30 minutes after the larvae were transfered to the auxin containing medium, with little inter-individual difference.
Before start
Genetically modified flies need to be generated before the present protocol can be implemented.
These flies should contain:
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a transgene allowing the expression of the auxin-dependent F-box protein TIR1, usually under the control of the UAS/Gal4 system (to achieve a spatial control of the degradation),
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the AID degron inserted (typically using CRISPR) to the coding sequence of the gene of interest.
See materials for an illustrative figure.
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Attachments
Steps
Carry out AID protein degradation in drosophila larvae
Initiate larvae production
Transfer in standard wheat cornmeal fly tubes about 50 healthy young adult flies (1/3 males, 2/3 females) per tube
Let the flies lay eggs at 25°C
Remove the flies from the tubes
Incubate the emptied tubes for 96h 0m 0s at 25°C until the wandering L3 Larvae appear
Preparation of NAA containing medium
Mix 1.2g of agar 8g of sucrose0.093g of NAA 200mL of water.
Microwave until complete agar melting
Cool down to 70°C
Pour 30mm plastic petri dishes (5ml of melted agar solution per dish)
Store in a plastic bag at 4°C protected from light
Triggering protein degradation in L3 larvae
Equilibrate NAA 30 mm dishes, and control dishes, prepared as described in section 2, to room temperature, and add a pinch of yeast granulates to the dishes
Using forceps, gently but quickly transfer up to 50 L3 larvae from tubes prepared as described in section 1 to a 30 mm dish containing NAA (same for control no NAA agar dish)
Quickly seal the dishes using a piece of parafilm
to prevent the larvae from escaping
Puncture tiny holes (small enough so that larvae cannot pass through) in the parafilm membrane using a needle in order to ensure oxygen and CO2 exchange
Incubate the dishes at 25°C the required amount of time
Remove the parafilm seal
Using forceps, gently collect the larvae from the agar dishes, and quickly proceed to desired operation, typically dissection of the imaginal discs.
