Table 1.
Summary of selection and counterselection markers
| Marker | Species origin | Encoded protein | Reference | Marker size (bp) | Drug(s)a | Solvent | Bacterial selection (μg/mL) | Drosophila size ESC/ECC (μg/mL) | Cost ($/vial) |
|---|---|---|---|---|---|---|---|---|---|
|
| |||||||||
| Resistance markers | |||||||||
| nptII | Klebsiella pneumoniae | neomycin phosphotransferase II | Davies and Smith, 1978 | 795 | kanamycin (bacteria), G418 sulfate (geneticin) | MQ H2O | 30 | 350 | 0.07 |
| pac | Streptomyces alboniger | puromycin N-acetyltransferase | Vara et al., 1985 | 600 | puromycin HCl | MQ H2O | 100 | 250–500 | 2.32– 4.64 |
| bsr b | Bacillus cereus | blasticidin S-resistance | Itaya et al., 1990 | 423 | blasticidin S | MQ H2O | 100 | 25–45 | 0.41– 0.73 |
| hph c | Escherichia coli | hygromycin B phosphotransferase | Gritz and Davies, 1983 | 1,026 | hygromycin B | MQ H2O | 75 | 35–45 | 0.04– 0.05 |
| ble d | Streptoalleteichus hindustanus | bleomycin resistance protein | Oliva-Trastoy et al., 2005 | 375 | zeocin or phleomycin | 1M HEPES | 25 or – | 350 | – or 9.35 |
| Sensitivity markers | |||||||||
| sr39TK e | herpes simplex virus-1 | thymidine kinase | Black et al., 2001 | 1,131 | ganciclovir | 0.1N NaOH | – | 4 | 0.01– 0.04 |
| FCU1 e | Saccharomyces cerevisiae | FCU1 | Erbs et al., 2000 | 1,122 | 5-fluorocytosine | 1×PBS | – | 10–15 | <0.01 |
The efficacy of seven different markers (five selection and two counterselection) were tested for in vivo selection and counterselection in Drosophila melanogaster. For each marker, we established an effective selection and counterselection concentration (ESC and ECC) defined as the minimal amount of drug required to either eliminate all non-resistant flies without significantly affecting resistance marker expressing fly viability or to eliminate all sensitivity-marker-expressing flies without significantly affecting non-sensitive flies. For the five selection markers, selection concentrations were also determined for use in bacteria.
Drugs used to determine cost were G418 sulfate (VWR 97063-060), puromycin HCl (VWR 97064-280), blasticidin S (VWR 71002-676), hygromycin B (VWR AAJ6068103), phleomycin (VWR AAJ67027-8EQ), ganciclovir (TCI America 50-155-694), and 5-fluorocytosine (TCI America 50-014-34810).
Although basal marker expression is enough to confer effective drug resistance when expressed from tested genomic docking sites, heat-shockenhanced expression is required when inserted into low-expressing genomic loci. Lower marker expression is also associated with blasticidin-S-related toxicity in resistant animals at higher concentrations (≥50 μg/mL).
Effective hygromycin B resistance requires heat-shock-enhanced expression in all tested contexts.
Zeocin was found ineffective at selecting flies. Phleomycin is a poorly effective selection agent, with high-ESC, batch-specific variability.
At higher concentrations, both tested counterselection drugs show general toxicity, with ganciclovir exhibiting toxicity above 15 μg/mL and 5-fluorocytosine above 50 μg/mL.