ABSTRACT
Transgenic mosquitoes generated by transposable elements (TEs) often poorly express transgenes owing to position effects. To avoid these effects, the ΦC31 site-directed recombination system was used to insert transgenes into a locus favourable for gene expression in Aedes aegypti. We describe phenotypes of mariner Mos1 TE and ΦC31 transgenic mosquitoes expressing the enhanced green fluorescent protein (EGFP) reporter in midguts of blood-fed females. Mosquitoes of nine TE-generated lines [estimated transformation frequency (TF): 9.3%] clearly expressed the eye-specific selection marker but only 2/9 lines robustly expressed the EGFP reporter. The piggyBac TE-generated ΦC31 docking strain, attP26, supported recombination with attB site containing donors at an estimated TF of 1.7-4.9%. Using a codon-optimized ΦC31 integrase mutant instead of the 'wild-type' enzyme did not affect TF. Site-directed recombination of line attP26 with an attB-containing donor expressing EGFP from the Ae. aegypti carboxypeptidase promoter produced one transgenic line with blood-fed females expressing the reporter in midgut tissue. Docking strain attP26 also supported robust expression of Flock House virus B2 from the Ae. aegypti polyubiquitin promoter. Our data confirm that eye-specific selection marker expression alone is not a reliable indicator for robust gene-of-interest expression in Ae. aegypti and that the ΦC31 system can ensure predictable transgene expression in this mosquito species.
Subject(s)
Aedes/metabolism , Bacteriophages , Gene Transfer Techniques , Transgenes , Animals , DNA Transposable Elements , DNA-Binding Proteins/metabolism , Female , Gastrointestinal Tract/metabolism , Gene Expression , Genes, Reporter , Integrases/metabolism , Promoter Regions, Genetic , RNA Interference , Recombination, Genetic , Transposases/metabolismABSTRACT
Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.
