Techniques for Identifying and Sorting Transgenic Mosquito Larvae.

Abstract

Transgenic mosquitoes are used in many aspects of mosquito research. First, they can help answer biological questions to advance scientific knowledge-for example, in the fields of mosquito-pathogen interactions, insect immunity, or olfaction. Second, transgenic technologies may be used to develop much needed novel vector control strategies, such as mosquitoes that are unable to transmit disease or transgenes that sterilize mosquito females to suppress vector populations. Here, we introduce how researchers use various selection markers to screen for transgenic mosquito larvae following a transgenesis experiment. Common procedures include using a binocular fluorescence microscope for initial screening. For higher-throughput screening, a flow cytometer known as Complex Object Parametric Analyzer and Sorter (COPAS) can be used to stabilize transgenic lines through the purification of homozygous individuals or to manage transgene frequency in established transgenic lines. In particular, COPAS sorting allows the production of mosquito larval cultures composed of a mixture of genotypes (control and genetically modified larvae) with the goal of raising both groups of mosquitoes under the same environmental conditions in preparation for a controlled phenotype assessment. It can also be used to produce large populations of male mosquitoes, which should facilitate the development of mosquito control intervention strategies similar to the sterile insect technique (SIT), which aims to release large numbers of sterile males that will mate with and sterilize wild females to suppress mosquito populations. Finally, the utilization of a puromycin resistance marker cassette to screen for transgenic Anopheles larvae is also introduced.