Table 2 Puromycin resistance cassette allows selection of transgenic larvae
Puromycin Concentration (µg/ml)
Larval Genotype0510204080
X1/X1 (no pac)17093*14**000
SG1/X1 (one copy of pac)n.a.16914513711651**
SG1/SG1 (two copies of pac)n.a.188183177168*80**
LRIM-G/X1 (one copy of pac)n.a.160150163130105**
LRIM-G/LRIM-G (two copies of pac)n.a.172179137152126**
DG2/+ (one copy of pac on piggyBac transposon)n.a.173177180179164*
  • The SG1, LRIM-G, and DG2 transgenic constructs carry the puromycin acetyltransferase (pac) gene under the control of the baculovirus OpIE2 promoter as a selection marker for transgenesis. Two hundred neonate larvae of each of the shown genotypes (heterozygous or homozygous pac) were distributed with the COPAS machine in 8 ml of demineralized water containing the indicated concentration of puromycin. Larvae were fed a small amount of finely ground TetraMin fish food daily. The number of surviving larvae was scored after 6 d of larval growth (although results were almost identical by 4 d after puromycin exposure). n.a. = not analyzed. Asterisks denote larvae that were visibly delayed in their development (*=moderate delay, **=strong delay). Note that 20 µg/µl of puromycin is sufficient for the full elimination of control larvae. The SG1 and LRIM-G transgenic constructs were prepared in the Gateway pattBRfB2 (File S2) and pDSAP transgenesis vectors, respectively, and inserted into the X1 docking locus, whereas the DG2 construct was prepared in a piggyBac plasmid whose integration site was mapped to a different locus (chromosome 3L: 3708611).