TITLE: Auxin-induced (AID) protein degradation in drosophila larvae 
AUTHORS: Denis Jullien, Adeline Payet, Emmanuelle Guillou, Henri-Marc Bourbon, Sandra Bernat-Fabre, muriel.boube-trey

[DESCRIPTION]
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:
1) 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),
2) 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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[GUIDELINES]
The method described here to induce targeted protein degradation with the AID system in drosophila larva is based on feeding the animal with auxin containing nutritive medium. Once present in the digestive asparatus, auxin penetrates in the tissues of the larva.

The protocol workflow is:

Day 1: young well-fed adults are transfered to fresh fly tubes.
Allow the flies to lay eggs for 15 hours, then remove the adults from the tube. This allow to get larvae all at a similar stage, and also avoid overcrowding. 

Day 3: prepare auxin containing petri dishes
30 mm plastic petri dishes containing a nutritive medium that contains auxin need to prepared.

Day 4 to day 5: collect larvae and trigger protein degradation, and proceed to phenotype analysis.
To trigger protein degradation, larvae are transfered using forceps to the auxin dishes.


[STEPS]
SECTION: Carry out AID protein degradation in drosophila larvae
1. Initiate larvae production


 

SECTION: Carry out AID protein degradation in drosophila larvae
2. Preparation of NAA containing medium
 

SECTION: Carry out AID protein degradation in drosophila larvae
3. Triggering protein degradation in L3 larvae
SECTION: Carry out AID protein degradation in drosophila larvae
1.1.  Transfer in standard wheat cornmeal fly tubes about 50 healthy young adult flies (1/3 males, 2/3 females) per tube
 

SECTION: Carry out AID protein degradation in drosophila larvae
1.2. Let the flies lay eggs    at 25 °C
SECTION: Carry out AID protein degradation in drosophila larvae
1.3. Remove the flies from the tubes
SECTION: Carry out AID protein degradation in drosophila larvae
1.4. Incubate the emptied tubes for 5760 min at 25 °C until the wandering L3 Larvae appear

SECTION: Carry out AID protein degradation in drosophila larvae
2.1. Mix 1.2 g of agar   , 8 g of sucrose  , and 0.093 g of NAA  , with 200 mL of water.
 
If required, depending on the nature of the negative control, prepare the same mixture without NAA to produce the control medium. Note that in flies expressing TIR1, a leaking degradation of the AID-tagged protein occures in the absence of auxin. 

SECTION: Carry out AID protein degradation in drosophila larvae
2.2. Microwave until complete agar melting
SECTION: Carry out AID protein degradation in drosophila larvae
2.3. Cool down to 70 °C

SECTION: Carry out AID protein degradation in drosophila larvae
2.4. Pour 30mm plastic petri dishes (5ml of melted agar solution per dish)
SECTION: Carry out AID protein degradation in drosophila larvae
2.5. Store in a plastic bag at 4°C protected from light
 

SECTION: Carry out AID protein degradation in drosophila larvae
3.1. 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
SECTION: Carry out AID protein degradation in drosophila larvae
3.2. 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)
SECTION: Carry out AID protein degradation in drosophila larvae
3.3. Quickly seal the dishes using a piece of parafilm  
to prevent the larvae from escaping

SECTION: Carry out AID protein degradation in drosophila larvae
3.4. 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
SECTION: Carry out AID protein degradation in drosophila larvae
3.5. Incubate the dishes at 25 °C the required amount of time
 


SECTION: Carry out AID protein degradation in drosophila larvae
3.6. Remove the parafilm seal
SECTION: Carry out AID protein degradation in drosophila larvae
3.7. Using forceps, gently collect the larvae from the agar dishes, and quickly proceed to desired operation, typically dissection of the imaginal discs.