Making recombined fly APL-GAL4

APL-GAL4 line is equal to VT43924-GAL4, UAS-GAL4.

The UAS-GAL4 line I used is: y[1] w[1118]; P{w[+mC]=UAS-Gal4.H}3A (http://flybase.org/reports/FBst0307136.html).

Actually, before recombination, I outcrossed VT43924-GAL4 and UAS-GAL4 lines to a wild-type background suitable for olfactory memory. I suggest you to outcross those VT lines because they are not in an isogenic background.

I list the procedure below:

Collect female virgin of either VT43924-GAL4 or UAS-GAL4. (I forgot which one I picked up, and it doesn't matter.)

Set up cross between virgins and the other line.  VT43924-GAL4 (III) X UAS-GAL4 (III)

Collect female virgin of VT43924-GAL4/ UAS-GAL4 (III)

Set up cross between ☿ VT43924-GAL4/ UAS-GAL4 (III) and ♂ TM3/ Sb (Before cross you might want to keep ☿ VT43924-GAL4/ UAS-GAL4 (III) flies in higher temperature (at least room temp.) for a while (at least one more week). It would help to increase recombination rate.)

Collect the male flies carrying TM3 marker with the darkest red eyes. ♂ VT43924-GAL4, UAS-GAL4/ TM3 (While collecting you can see some while-eye flies. Theoretically one white-eye fly means one recombined fly.)

(Be careful for collecting the "darkest" red-eye flies. I had literally continued this step for more than one week to get a lot of candidates, and then pick up the darkest one.)

Set up cross between ♂ VT43924-GAL4, UAS-GAL4/ TM3 and ☿ TM3/ Sb

Collect ♂ and ☿ VT43924-GAL4, UAS-GAL4/ TM3 in the same vial. They are supposed to mate with each other.

Collect homozygous VT43924-GAL4, UAS-GAL4 (III) flies. Done!

I never do any PCR to verify the recombination but phenotypic selection.

I have made a cross between UAS-GAL4 and UAS-mCD8::GFP; UAS-mCD8::GFP (5137; 5130) to make sure there is no noticeable leaky expression from this UAS-GAL4 line. However, when I recently used the Janelia Farm reporter (ex: 32194 w[*]; P{y[+t7.7] w[+mC]=20XUAS-IVS-mCD8::GFP}attP2), which incorporate more copies of UAS and Myosin heavy chain intron16 (Pfeiffer et al., 2010), I found extra expression pattern under the control of APL-GAL4, as compared to the old version reporter 5137; 5130. Before you really start the recombination, you might want to confirm the leaky expression level of UAS-GAL4 line with Janelia Farm reporter.

If you want to make balanced flies incidentally, you can use CyO/ Sp; TM3/ Sb to replace all the TM3/ Sb flies above.