Selecting Transgenic Mosquito Larvae with Puromycin

The selection of mosquito transgenic larvae using a nonfluorescent approach can be advantageous to reserve fluorophores for downstream applications, such as immunostaining or for the study of promoter activity by cloning a fluorescence reporter gene under the control of that promoter. We previously reported that puromycin selection is efficient in transgenic Anopheles gambiae or Anopheles coluzzii larvae expressing an OpIE2-pac selection marker. A concentration of puromycin of >10 µg/mL is lethal for Anopheles larvae, unless they carry the resistance gene, conferring them resistance to puromycin concentrations of 25-80 µg/mL. A drawback of this fully dominant selection marker is that, unlike with fluorescence markers, homozygous transgenics cannot be distinguished from heterozygotes. Here, we outline the procedure for selecting puromycin-resistant transgenic Anopheles larvae.