Dominant lethal Aedes aegypti mosquito

A modified strain of the Aedes aegypti mosquito, designated as OX513A(My1), was developed to exhibit dominant lethality in both males and females when reared in the absence of tetracycline and includes a red fluorescent protein (DsRed2) as a visible marker.

In the presence of tetracycline, the synthetic tetracycline‐transcriptional activator (tTAV) variant preferentially binds tetracycline instead of the tetracycline operator, thus transcription is repressed and occurs at a basal level. In the absence of tetracycline, 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.

This approach is similar to sterile insect technique, which uses radiation to produce sterile males, but does not incur great fitness reductions caused by the radiation required to produce sterile males. Modified mosquitoes are reared in the presence of tetracycline, which allows for full development. Upon release, mating with the modified mosquitoes results in lethality in the next generation.

Initial transformation: Aedes aegypti Rockefeller strain
Backcrossing: Ae. aegypti Latin strain (from Instituto Nacional de Salud Publica, Mexico)

pLA513 and phsp-pBac

The pLA513 plasmid was co-transformed with the phsp-pBac helper plasmid, which served as a source for the piggy Bac transposase.

Transcription of the DsRed2 protein begins at the Drosophila melanogaster actin 5c promoter and terminates at the D. melanogaster dorsomycin 3' untranslated region. The promoter drives expression of the fluorescent protein marker, which causes an accumulation of soluble protein within the cells.

Transcription of the tetracycline‐transcriptional activator variant (tTAV) begins at the D. melanogaster heat shock protein 70 promoter and terminates at the D. melanogaster DNA-binding protein K10 3' untranslated region (poly-adenylation signal). The transcript initially includes a D. melanogaster alcohol dehydrogenase intron at the 5' end of the transcript to enhance expression of tTAV. Immediately adjacent to the tTAV cassette is a tetracycline operator, which 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. Thus, under these conditions, transcription of tTAV is expected to be strong and with production of the tTAV protein occurring at elevated levels.

Please note:
- tTAV is a synthetic construct and contains sequences from the Escherichia coli tetracycline repressor and the Human herpesvirus 1 viral protein 16 transactor
- DNA deletions in the piggyBac sequences prevent mobility of the transposon. Additionally, the construct does not introduce a transposase. Thus, re-mobilization is not expected.
- Southern blot analysis confirmed a single insertion into the mosquito genome occurred
- Inverse PCR and sequencing suggested that the insertion does not interrupt an open reading frame
- The plasmid backbone contains an ampicillin resistance gene, which was not detected in the transformants

Modified mosquitoes can be detected by red fluorescence under yellow light (583 nm) due to the DsRed2 protein.

The D. melanogaster alcohol dehydrogenase intron is spliced out of pre-mRNA, and thus also allows the distinction between cDNA (transcripts) and genomic DNA during RT-PCR analysis.

Information on OX513A(My1)

OX513A(My1) is a bisex RIDL strain, which means that both female and male insects die unless supplied with the supplement, which in the case of OX513A(My1) is the antibiotic tetracycline.

Released bisex RIDL insects and their progeny die within a few weeks so releases must be sustained to maintain the control.

Source: Oxitec (see developer field above).

Information on the Release of Insects carrying a Dominant Lethal (RIDL) technology

Release of Insects carrying a Dominant Lethal (RIDL) is a method using recombinant DNA technology to create genetically modified insects for biological control. The dominant lethal gene kills the insects but it can be repressed by an external additive, which allows the insects to be reared in manufacturing facilities. This external additive is commonly administered orally, and so can be an additive to the insect food. The insects can also be given genetic markers, such as fluorescence, that make monitoring the progress of eradication easier.

There are potentially several types of RIDL, but the more advanced forms have a female-specific dominant lethal gene. This avoids the need for a separate sex separation step, as the repressor can be withdrawn from the final stage of rearing, leaving only males.

These males are then released in large numbers into the affected region. The released males are not sterile, but any female offspring their mates produce will have the dominant lethal gene expressed, and so will die. The number of females in the wild population will therefore decline, causing the overall population to decline.

Using RIDL means that the males will not have to be sterilized by radiation before release (as done with the "Sterile Insect Technique" (SIT) using radiation), making the males healthier when they need to compete with the wild males for mates.

Source: Wikipedia (see link below).