Electroporation-mediated nucleic acid delivery during non-embryonic stages for gene-function analysis in Anopheles...

The delivery of exogenous nucleic acids to eggs or non-embryonic individuals by microinjection is a vital reverse genetics technique used to determine gene function in mosquitoes. However, DNA delivery to eggs is complex and time-consuming, and conventional, non-embryonic-injection techniques may result in unobvious phenotypes caused by insufficient absorption of nucleic acid fragments by cells at target body parts or tissues. In this study, we developed a set of electroporation-mediated non-embryonic microinjections for the delivery of exogenous nucleic acids in Anopheles sinensis. Gene silencing using this method led to down-regulation of target gene expression (AsCPR128) by 77% in targeted body parts, compared with only 10% in non-targeted body parts, thus increasing the defect-phenotype rate in the target area by 5.3-fold, compared with non-shock injected controls. Electroporation-mediated somatic transgenesis resulted in stable phenotypic characteristics of the reporter gene at the shocked body parts during the pupal-adult stages in about 69% of individuals. Furthermore, injecting plasmid DNA near the ovaries of female mosquitoes after a blood meal followed by electric shock produced three germline G₁ transgenic lines, with a transformation rate of about 11.1% (calculated from ovulatory G₀ females). Among the positive G₁ lines, 42%, 40%, and 31% of individuals emitted red fluorescence in the larval stage. When the red fluorescent larvae developed into adults, green fluorescence was emitted from the ovaries of the females upon feeding. These results suggest that electroporation-mediated non-embryonic microinjection can be an efficient, rapid, and simple technique for analyzing gene function in non-model mosquitoes or other small insects.