Starter strains
Following two strains were used for stock establishment.
Starter transposon strain:
DGRC # 109787
w; P{w+mC=PBac{EGFP-IV}KM0007}KM0007}f1m1
The piggyTrap transposon, PBac{EGFP-IV}, was introduced by the P-element vector, pCaSpeR4. PBac{EGFP-IV} and its flanking sequences were derived from the piggyTrap insertion in the tna gene (see
DGRC#109723). The P-element insertion site is unknown.
Transposase strain:
DGRC # 109788
w; P{w+mC=Hsp83-PBac-ORF}2 P{w+mC=Hsp83-PBac-ORF}5
piggyBac-transposase is expressed under the Hsp83 promoter. At least two copies on the 3rd chromosome.
Screening and stock establishment
Above two strains were mated with each other. The progeny was allowed to mate and lay eggs on a plate. Embryos were dechorionated by bleach and washed with water. Embryos were placed in heptane and transferred on glass slides with a pasteur pipette. Because embryos sink to the bottom in heptane, embryos spread to a single layer on the glass slide. After brief evaporation of heptane, embryos were covered with mineral oil for avoiding desiccation, and screened under the fluorescence stereo microscope. GFP fluorescent embryos were picked and transferred to usual vials with food and allow to develop to adult stage. An individual fly emerged was mated to w flies for establishing a stock. In each generation, fluorescent female flies were selected and backcrossed to w males repeatedly until fixed to white-eyes. In general only strains that show fluorescence at adult stage were kept. Some strains were screened at the stage of larvae and imagoes. Some strains were hard to separate the mini-w marker on the P-element from the piggyTrap insertion. In this case P-elements were excised by crossing to delta2-3 leaving behind the piggyTrap insertion causing fluorescence.
The insertion sites were determined by the conventional inversePCR (iPCR) technique using following primers.
For iPCR amplifications,
GFP.U2 (5'-CCGCTACCCCGACCACATGAAG-3')
GFP.R2 (5'-AGGGTCAGCTTGCCGTAGGTG-3')
In the case of poor amplification, 2nd nested PCR was performed using following primers,
GFP.U1(5'-AGCACGACTTCTTCAAGTCCGCCAT-3')
GFP.R1 (5'-ACGCTGAACTTGTGGCCGTTTACGT-3')
Sequence reaction was performed using a primer,
GFP5'.R (5'-ACCACCCCGGTGAACAGCT-3')