Genetic and PCR analyses of targeting candidates of crb∷mEosFPKI and dArf6KO

Targeted alleleTargeting candidatesRpr testFRT+loxP+X chrPCR verified
crb∷mEosFPKINon-third chr candidatesRpr+ 12217/122122/1220/122ND
Rpr[−] 30/33/30/3ND
Third chr candidatesRpr+ 30/33/32a/3
Rpr[−] 110/1111/117/11
dArf6KObNon-second chr candidatesRpr+ 12054/120ND0/120ND
Rpr[−] 20/2ND0/2ND
Second chr candidatesRpr+ 20/2NDND
Rpr[−] 0
  • Rpr+, scored by lethality or strong wing phenotypes in the presence of Gal4221[w−] or Gal4477[w−]. The total number of Rpr[−] false positives of crb∷mEosFPKI can be estimated as 10 [6 from non-third chromosome candidates (3 × (256/125)], plus 4 from third chromosome candidates). FRT+ or loxP+, scored by eye color variegation in the presence of constitutively expressed FLPase or Cre recombinase. Approximately 87% [(263 − (17 × 2))/263] of crb∷mEosFPKI false positives and ∼57% [(124 − 54)/124] of dArf6KO false positives showed damaged FRT sites. X chr, candidates that were mapped to the X chromosome. Here, none of the nontarget chromosome false positives were mapped to the X chromosome. Since the 4th chromosome is extremely small, therefore unlikely to harbor any nonspecific targeting events, “X chr” data indicate that virtually all of the nontarget chromosome false positives retained their donor DNA on the original chromosome, due to either damaged FRT sites or insufficient excision of donor DNA. ND, not done; —, not applicable.

  • a Tandem insertion mutants.

  • b Only dArf6KO candidates recovered from screening crosses with regular Pin/CyO balancer stock are listed here (see Table 2).