Generating Somatic Clones by FRT Recombination

The following protocol has been adapted from one made by Tian Xu. Tian introduced two tandem yeast FRT DNA elements near the centromere of each chromosomal arm. These can mediate high frequency mitotic recombination upon expression of the yeast FLP recombinase. Mosaic clones for mutations located distally to the FRT elements are identified using a mosaic cell-autonomous marker. Adult markers that are available are white for eye tissue, yellow for cuticle and bristles, and Sb for bristles. Markers for all stages and all tissues include proteins tagged with the MYC epitope, arm-lacZ, and GFP. The system is designed such that mosaic clones are identified by their lack of a particular marker. The following FRT stocks are used for recombination:

  • X: P{neoFRT}18A
  • 2L: P{neoFRT}40A
  • 2R: P{neoFRT}42D
  • 3L: P{neoFRT}80B
  • 3R: P{neoFRT}82B
Recombining Mutation On FRT Chromosome
  1. Cross mutant strain to a w- ; P{white-un1}, P{neoFRT} stock.
  2. Mate ten w-/w+ ; P{white-un1}, P{neoFRT} /mutation heterozygous virgin females to a w- balancer stock in vials. Allow to premate for two days, then mate at 25C in vials with G418-treated food for 24 hours. Transfer adults to fresh G418-treated food after 24 hours; repeat several times.

    The dose of G418 to give varies with the FRT insert (probably because of position effects on NeoR gene expression). Melt food in microwave and add following volumes of 25 mg/ml G418 to 10 ml of food:
    • P{neoFRT}18A: 0.20 ml
    • P{neoFRT}40A: 0.15 ml
    • P{neoFRT}42D: 0.15 ml
    • P{neoFRT}80B: 0.10 ml
    • P{neoFRT}82B: 0.20 ml
    Mix the G418 into the food evenly. Raise at 25C. The life cycle is retarded somewhat by the G418.

    NB: These dosages enrich but do not select for the FRT. Too high a dose and everything dies.
  3. Select w- males. These are also resistant to G418, and thus have had a crossover between P{white-un1} and P{neoFRT}. Since the endogenous white gene is located far away from the P{neoFRT} insert on the X chromosome, there is a chromosome containing Bar on the P{neoFRT} chromosome. Select for recombinant males that have the P{neoFRT} but are non-Bar. The recombination distances between the P{neoFRT} and P{white-un1} inserts are estimated to be:
    • P{neoFRT}18A to Bar: 4 map units
    • P{neoFRT}40A to P{white-un1}30C: 20 map units
    • P{neoFRT}42D to P{white-un1}47A: 5 map units
    • P{neoFRT}80B to P{white-un1}70C: 7 map units
    • P{neoFRT}82B to P{white-un1}90E: 15 map units
  4. Select several recombinant males and mate individually to a w- balancer stock and then serially testcross the males individually to females carrying your mutation. Establish balanced stocks of those lines in which the testcross shows noncomplementation between the recombinant chromosome and the chromosome carrying your mutation.
Making Mosaic Flies
  1. Cross the w- ; mutation P{neoFRT} stock to the appropriate w- FLP ; marker P{neoFRT} stock. Allow to premate for two days. Collect eggs for 12 hours at 25C and let them age for a further 24 hours. Most animals will be first instar larvae.
  2. For hsp-FLP induced clones, incubate F1 larvae at 38C for 60 minutes, and then return to 25C until they are analyzed. About 90% of the eyes should carry mosaic clones under these conditions. Without exposure to the 38C shock, fewer than 1% of the eyes contain mosaic clones.

original text by Richard Carthew