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Selecting Transgenic Mosquito Larvae with Puromycin.

¹CNRS UPR9022, INSERM U1257, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, 67000 Strasbourg, France e.marois@unistra.fr.

Cold Spring Harbor Protocols

|September 11, 2023

Summary

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Puromycin selection offers a nonfluorescent method for identifying transgenic mosquito larvae, preserving fluorophores for other uses. This approach efficiently selects for resistant larvae, though it doesn't distinguish heterozygotes from homozygotes.

Area of Science:

Medical Entomology
Molecular Biology
Genetics

Background:

Nonfluorescent selection methods for transgenic mosquito larvae are valuable for preserving fluorophores for downstream applications like immunostaining or promoter activity studies.
Puromycin selection using the OpIE2-pac marker has been previously shown to be effective in transgenic Anopheles gambiae and Anopheles coluzzii larvae.
A puromycin concentration above 10 µg/mL is lethal to wild-type Anopheles larvae, while transgenic larvae carrying the resistance gene can tolerate concentrations of 25-80 µg/mL.

Purpose of the Study:

To outline a detailed procedure for the selection of puromycin-resistant transgenic Anopheles larvae.
To provide an alternative nonfluorescent selection strategy for generating transgenic mosquitoes.

Main Methods:

Utilizing the OpIE2-pac selection marker for puromycin resistance in transgenic Anopheles larvae.

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Administering lethal concentrations of puromycin (>10 µg/mL) to distinguish resistant transgenic larvae from susceptible wild-type larvae.

Establishing puromycin resistance levels for transgenic Anopheles larvae ranging from 25-80 µg/mL.

Main Results:

The puromycin selection procedure effectively identifies transgenic Anopheles larvae.
This method allows for the selection of transgenic mosquito larvae without the use of fluorescent markers.
A limitation of this dominant marker is the inability to differentiate between homozygous and heterozygous transgenic individuals.

Conclusions:

Puromycin selection provides an efficient, nonfluorescent method for isolating transgenic Anopheles larvae.
This technique is advantageous for conserving fluorescent markers for subsequent experimental analyses.
The described procedure facilitates the generation and selection of transgenic mosquito lines.