USEFUL STOCKS#

• yw UAS-mCD8::GFP; S/CyO; +
• 10XUAS-mCD8::GFP; + ; TM2/TM6B

Step 1A. Screening hemidriver combinations #

Combine each AD and DBD combination together with UAS-GFP. The goal here is to visualize the expression pattern of this hemidriver combination. The result is not a stable stock, however, so this is for screening purposes only. Here it is convenient to begin with virgins having UAS-GFP on the X chromosome and double balanced on the second chromosome.

Step 1B. Screening hemidriver combinations using GDBD + GFP#

This is the same as Step 1A except you combine virgins having UAS-GFP on the X chromosome and double balanced on the third chromosome. This can be convenient if you are crossing one GDBD to many AD hemidrivers.

Step 1C: Screening hemidriver combinations where the AD already has a GDBD#

Sometimes lines from Bloomington will already have an GDBD attached to the AD you want to combine to another hemidriver. Below are the steps on how to get rid of the unwanted GDBD using virgins that UAS-GFP on the X chromosome and double balanced on the second chromosome.

Step 1D: Screening hemidriver combinations where the GDBD already has an AD#

Sometimes lines from Bloomington will already have an AD attached to the GDBD you want to combine to another hemidriver. Below are the steps on how to get rid of the unwanted AD using virgins that UAS-GFP on the X chromosome and double balanced on the third chromosome.

Things to note for Step 1 & common errors:

• Watch out for presence of different phenotypic markers and when you should/should not see certain ones + what each marker represents (i.e eye color, wings, TM6B or TM2 etc)
  • Double check the genotype of the final fly, make sure that expected phenotype matches the actual phenotype
  • Gender of the flies matter because there is no recombination in male flies so be careful when planning and setting up crosses
• Make sure the females used to set up the crosses are virgins
• Be strategic about planning crosses - if you are crossing one AD to many GDBD stocks you might want to cross that AD to the GFP stock rather than crossing each GDBD stock and vice versa
• It is important to time the eclosion of your hemidriver x GFP cross to the eclosion of your other hemidriver stock so that you can virgin and collect males at the same time to start the next cross quickly

Step 1E: Screening individual hemidrivers

Note that this step can proceed in parallel with Step 1A. Step 1E is optional, and we often omit it. However, it might make sense to include this step if you have one or more hemidrivers that you plan to test with lots of other partners. For example, if you have just two potentially good AD hemidrivers, and you plan to test each of these with ten different DBD partners, it’s probably a good idea to pre-screen the two AD hemidrivers; that say, if either AD is a dud, there’s no point in testing it with all those DBD partners. To expedite the whole process, you would want to set up the crosses with the DBD partners even before you get the results of the AD screening – with the idea that you would trash those AD x DBD crosses if the AD is a dud.

Screening AD lines: 

Screening DBD lines: 

Step 2. Making a stable split-Gal4 line   #

If any of your hemidriver combinations look good, then you should stabilize that combination. First you need to double-balance each of your hemidrivers:

Then you use these stocks to generate a stable combination (i.e., a stable split-Gal4 line):