Make a fly stock which is homozygous for a mutant allele of gene X and which is homozygous white minus. We use the null allele of white, w-1118. Obviously if the mutant allele of gene X is lethal, it has to be balanced over a chromosome with a dominant marker.
The strategy is to put the chromosome with mutant gene X in trans to the chromosome containing the white gene. Recombination events between sister chromosomes during mitosis are induced by X irradiation. If recombination occurs between the centromere and the white gene it will produce a daughter cell that is homozygous mutant for white and gene X. Replication will expand cell numbers to the point that in the eye a white patch is seen in a red eye.
Make a mass fly cross between w-; X- and w-; P[w+]. I prefer to mate 400 of each genotype putting 100 pairs in one bottle. Incubate 24 hours at 25°C. Transfer flies to fresh bottles. These can either be egg laying bottles or regular bottles. I prefer egg laying bottles because the parameters for irradiation are more consistent. If you use egg laying bottles, use plates containing yeast-glucose medium (see end of section). Collect embryos for 12 hours at 25°C. Repeat collection. Incubate plates or bottles for a further 42 hours at 25°C. The flies will now be 48 +/-6 hours old and will be late first instar larvae. Inducing recombination at this time gives a balance between reasonable clone size and frequency of clones (about 1 in 20 flies). If you want to generate larger clones, irradiate at an earlier time, but be prepared to generate clones at a lower frequency.
Irradiate tlarvae in plates or unstoppered bottles with 1000 rads (115 kV, 5 mA). If using egg-laying plates, cut the plates in halves or quarters and place each piece in a bottle. When the flies have eclosed, they can be screened for clones. Most white clones appear black under a dissecting microscope. Fix and section the eyes as described in Section 19. However, before the flies are sacrificed, shine a bright light on them for a few minutes. This causes the pigment granules in the photoreceptor cells to migrate apically and makes it easier to score for white pigment in cells R7 and R8. Cut 1 to 4 micron thick sections and collect all of the sections serially. This is important as pigment may only be visible in R7 and R8 cells for one or a few sections. Do not stain the eye sections but mount in DPX and view under phase contrast. Pigment granules appear as black specks adjecant to each cell's rhabdomere.
Recipe for Yeast-Glucose Plates:
10% dry yeast
10% glucose
1.6% agar
3/100 (v/v) 10% tergocept
Boil water and add solids. Boil for 5 minutes while stirring. Pour into 15x60 mm
plates.