2nd Generation Friendly™ Aedes aegypti

Aedes aegypti mosquitoes were modified female-specific lethality for population suppression and red fluorescence for visual detection.

Sex-specific lethality is achieved through the use of alternative splicing and the expression of a synthetic tetracycline‐transcriptional activator (tTAV) variant. In females, splicing occurs such that the tTAV coding sequence is translated. In males, alternative splicing causes several stop codons to be included upstream of the tTAV coding sequence, interrupting translation and preventing expression of tTAV. In the presence of tetracycline (rearing conditions), tTAV preferentially binds tetracycline instead of the tetracycline operator, thus transcription is repressed and occurs at a basal level. In the absence of tetracycline (environmental conditions), tTAV binds the operator sequences to promote high levels of transcription. High levels of tTAV expression is toxic as it prevents the cells from producing other transcripts required for normal functioning and results in lethality. Thus, females can be produced in rearing conditions, but fail to develop in environments without the presence of tetracycline. Due to the alternative splicing mechanism, males carry and transmit the self-limiting genes to progeny without requiring tetracycline. Population suppression is thus achieved through the biased selection of male offspring.

The modified mosquitoes also include the Discosoma sp. DsRed2 protein for red fluorescence under yellow light for visual detection of modified organisms. In comparison to OX513A mosquitoes (see "Related LMOs"), a synthetic nuclear localization signal was included to localize DsRed2 to the nuclei of cells, improving visualization (fluorescence) in larvae and adult mosquitoes.

pOX5034

Fluorescent marker
Transcription of the fluorescent marker commences from the Autographa californica multiple nucleopolyhedrovirus immediate-early-1 gene promoter and terminates at the Simian vacuolating virus 40 (SV40) poly-adenylation signal. The transcript initially contains a Drosophila melanogaster scraps intron, two synthetic nuclear localization signals (nls) and the Discosoma sp. DsRed2 fluorescent protein. The scraps intron improves mRNA stability and is required for translation of the protein, but is not expected to be included in the translated polypeptide. After splicing and translation, the nls allows DsRed2 to transit and accumulate in the nuclei of cells.

Sex-specific lethality
Transcription of the Escherichia coli tetracycline-controlled transactivator (tTAV) commences from the D. melanogaster heat shock protein 70 minimal promoter and terminates at the SV40 poly-adenylation signal. The transcript contains the Aedes aegypti sex-specific intron, D. melanogaster ubiquitin and tTAV.  In females, splicing removes the sex-specific intron. The remaining transcript is translated to produce a poly-peptide containing ubiquitin and tTAV. Post-translation, ubiquitin is removed, leaving the functional tTAV. Ubiquitin is expected to enhance protein expression within insect cells. In males, splicing occurs from a further downstream site within the sex-specific splicing module. The resulting transcript then contains several stop codons upstream of ubiquitin and tTAV, which disrupt translation and thus prevents the production of the tTAV protein.

Transcriptional expression is also influenced by the tetracycline operator, which is adjacent to the tTAV cassette and acts as a repressible switch. In the presence of tetracycline, tTAV preferentially binds tetracycline rather than the operator sequences. Thus, transcription remains at a basal level and repressed. In the absence of tetracycline, tTAV binds the operator sequences and stimulates transcription, resulting in elevated levels of the tTAV protein.

Modified mosquitoes can be detected by red fluorescence under yellow light (583 nm) due to the presence of DsRed2 protein. Fluorescence is expected to be localized to the nuclei of cells.

Male mosquitoes contain insecticide-susceptibility genes, which are expected to dilute insecticide resistance in wild populations.

In Portuguese, the modified mosquito is known as "Aedes do Bem™".