Successful insertion and expression of a tetracycline transactivator in Anopheles stephensi associated with increased egg production and decreased hatching rate
Bibliographic record
Abstract
BACKGROUND: has pioneered production of aseptic, purified, vialed cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ) as vaccines and for controlled human malaria infections. More than 3500 individuals have received more than 9700 injections of PfSPZ worldwide. The PfSPZ are manufactured in aseptically reared female Anopheles stephensi mosquitoes. Since PfSPZ vaccines are intended primarily for some of the most disadvantaged people in the world, keeping costs low is imperative. One approach to reducing the cost of goods is to eliminate male mosquitoes from the production process, thereby doubling the numbers of PfSPZ-producing mosquitoes per unit space. We intend to do this by creating An. stephensi with a male-lethal allele controlled by the tetracycline conditional gene expression system. METHODS: Herein, we report the first step in this process, the creation of a driver line that expresses the reverse tetracycline transactivator (rtTA). RESULTS: After suboptimal results using the bZip early embryonic promoter, we produced three mosquito driver lines that expressed rtTA from three different genomic loci under the early embryonic vasa promoter. Expressing the rtTA under the vasa promoter significantly increased rtTA mRNA levels compared with under bZip. We were unable to achieve homozygosity in two of these lines even after 26 generations. In a third line we observed seven distinct insertions, six of which, including one located in an intron of a protein-coding gene, were homozygous. This line achieved homozygosity after being passed through seven generations, suggesting that the insertions did not disrupt the function of any crucial genomic locus. The levels of rtTA mRNA expression in the homozygous-viable line were higher than those in the other two lines, reinforcing the idea that the inability to reach homozygosity was not due to rtTA expression levels but rather the position of the insertion. The homozygous-viable line produced ~18% more eggs per female, and a hatching rate of larvae from eggs was 39% lower than that of wild-type An. stephensi. The next step will be to cross the driver line with an effector line containing a male-linked lethal gene regulated by the tetracycline responsive element (TRE).
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".