2. Dengue Fever
• Mosquito-borne viral disease
• Aedes aegypti = principal vector
• Estimated 390 million infections per year; 96 million
which manifest clinically (W.H.O Jul., 2016)
• Estimated 3.9 billion people, in 128 countries, are at
risk of infection (W.H.O Jul., 2016)
• No vaccine or therapeutic drug (available on market)
• Control at the level of the mosquito
• Bed nets ineffective (day-biting)
• Spraying and source reduction
• New approaches and vector control tools needed
3. Sterile Insect Technique (SIT)
• Release of overwhelming numbers of sterile
insects
• Environmentally friendly
• Species-specific population control
• Sterile insects compete for mates
• Mating with sterile insects produces no offspring
• Population reduction in the following generation
• Aedes aegypti is highly suitable for SIT
http://igtrcn.org/insect-genetic-technologies-to-control-blow-flies/
4. Problems of SIT against mosquitoes
• Fitness costs associated with irradiation
• SIT relies on the introduction of random lethal mutations
• Male-only release preferred
• Adult mosquitoes are likely to suffer damage during transit/release
5. Mitigating problems of SIT
• Hypothesized that insects could be engineered to carry a female-
specific, repressible, dominant lethal genetic system (release of
insects carrying a dominant lethal [RIDL]), which acts late in
development
• Removes need for irradiation-sterilization
• Allows removal of females prior to release
• Allows mass rearing in lab
• Allows density-dependent competition
• Previous mathematical modeling supports late-acting lethal
• Previous repressible lethal strains using “tet-off” system
6. AeAct-4 gene
• Expressed in female pupae indirect flight muscles (IFMs)
• q-PCR reveals low-abundance transcript in males
• Sex-specific alternative splicing may reduce amount of functional AeAct-4
• Use the promoter of this “female-specific” gene to drive
expression of a reporter gene in transgenic Aedes aegypti
7. AeAct4-DsRed
• Strong fluorescence in the IFMs of
both female pupae and adults
• Tissue-specific expression
• Supports use of AeAct-4 regulatory
sequences to drive expression of a
repressible deleterious or lethal
system in Ae. aegypti
8. Late-acting lethality
• IFMs are non-vital in Drosophila melanogaster
• Predicted disruption of IFMs in Aedes aegypti would result in a
flightless phenotype, rather than lethality
• However, likely to be lethal in the field (emergence, predation, locating blood
meal, and locating mate)
• AeAct-4 regulatory DNA provides one of the last opportunities in
development to kill females before blood-feeding
• Goal: Use the AeAct-4 gene regulatory sequences to drive the expression
of a repressible RIDL trait.
9. AeAct4-mediated marker expression
• Constructed seven transgenic AeAct4-tTA (OX3545) lines
• tTAV = tetracycline-repressible transactivator variant
• Encodes tetracycline-repressible transactivator (tTA)
X
tTA
• Crossed to a transgenic tRE-DsRed line (dual transgene hybrid)
• DsRed under control of a tTA responsive element (tRE)
• Multiple copies of tetracycline operator (tetO) + minimal promoter
10. AeAct4-mediated marker expression
X
tTA
DsRed
• Constructed seven transgenic AeAct4-tTA (OX3545) lines
• tTAV = tetracycline-repressible transactivator variant
• Encodes tetracycline-repressible transactivator (tTA)
• Crossed to a transgenic tRE-DsRed line (dual transgene hybrid)
• DsRed under control of a tTA responsive element (tRE)
• Multiple copies of tetracycline operator (tetO) + minimal promoter
11. AeAct4-mediated marker expression
X
tTA
Tc
• Constructed seven transgenic AeAct4-tTA (OX3545) lines
• tTAV = tetracycline-repressible transactivator variant
• Encodes tetracycline-repressible transactivator (tTA)
• Crossed to a transgenic tRE-DsRed line (dual transgene hybrid)
• DsRed under control of a tTA responsive element (tRE)
• Multiple copies of tetracycline operator (tetO) + minimal promoter
12. AeAct4-mediated marker expression
X
tTA
Tc
• Constructed seven transgenic AeAct4-tTA (OX3545) lines
• tTAV = tetracycline-repressible transactivator variant
• Encodes tetracycline-repressible transactivator (tTA)
• Crossed to a transgenic tRE-DsRed line (dual transgene hybrid)
• DsRed under control of a tTA responsive element (tRE)
• Multiple copies of tetracycline operator (tetO) + minimal promoter
13. AeAct4-mediated marker expression
• All AeAct4-tTA ; tRE-DsRed progeny
displayed strong red fluorescence in
the developing IFMs of late 4th instar
female larvae, as well as pupae and
adults
• Expression repressible by Tc
• Equivalent insects reared in medium
containing 30µg/mL Tc showed no
fluorescence
14. AeAct4-mediated marker expression
• Unexpectedly, sex-specific
alternative splicing did not always
prevent the expression of tTA in
males
• Expression of DsRed in the IFMs of
males when certain lines of AeAct4-
tTA were crossed with tRE-DsRed
15. AeAct4-mediated marker expression
• AeAct4-mediated expression of
tTA in dual transgene hybrid
mosquitoes produces the tTA
transactivator that then binds the
operator and triggers expression
of a reporter gene product
• What about expressing a lethal
effector gene?
16. AeAct4-mediated effector expression
• Induce flight-less phenotype using two separate transgenes
• AeAct4-tTA (OX3545) crossed to transgenic lines possessing lethal effector
genes
• Nipp1Dm (OX3547)
• Nuclear inhibitor of protein phosphatase 1
• michelob_x (OX3582)
• Inhibitor of apoptosis protein antagonist
• Both under tRE control
X
Nipp1Dm / michelob_x
OX3547/OX3582
17. AeAct4-mediated effector expression
• 69.8-98.3% of females with tTA+effector were flightless when raised in the absence of Tc
• Result reveals that expression of a suitable effector in the IFMs can cause a flightless
phenotype
• tTA can be used as the effector molecule in a repressible lethal system
18. AeAct4-mediated effector expression
• Observed flightless females in absence of effector gene
• Reasoned that the observed flightless phenotype of insects expressing tTA was due to VP16,
the domain which causes cellular toxicity
• Additional expression of VP16 should thus increase penetrance
• Penetrance = the proportion of individuals carrying a particular allele that also express the associated phenotype
19. Refining female-specificity and penetrance
• Utilized alternative splicing observed in native AeAct-4 RNA
• Start codon (ATG) introduced adjacent to 5’ end of male-specific exon
20. Refining female-specificity and penetrance
• Female-specific splice variant ATG is in frame with tTAV
• Male-specific splice variant ATG causes a frame shift, resulting in multiple stop codons
• Predicted to prevent the expression of functional tTA protein
21. Refining female-specificity and penetrance
• Constructed OX3604C
• Uses a single transgene
(combines both components
of the expression system)
• Driver component
• Effector component
22. Refining female-specificity and penetrance
• Crossed OX3604C with
tRE-DsRed
• Reared in absence of Tc in
order to analyze expression
of tTA
X
23. Refining female-specificity and penetrance
• Unlike some earlier lines, OX3604C did not drive expression of DsRed
in males
• RT-PCR for transgene products showed that although males did produce
RNA, transcripts encoding functional tTA were only present in females
24. Refining female-specificity and penetrance
• OX3604C mosquitoes reared in the absence of Tc showed a highly
penetrant, dominant, female-specific (see table S1) flightless phenotype
• When reared in presence of Tc only 0.3% of females showed flightless phenotype;
not significantly different from WT (p = 0.765)
26. Conclusions
• It is possible to engineer late-acting, repressible, tissue-specific,
and female-specific transgene expression to cause a flightless
phenotype in Aedes aegypti
• Flightless phenotype can be considered equivalent to lethality
• Inability to produce wing oscillation song
• Allows any life-stage mosquito to be released
• Tolerance of Ae. aegypti eggs to desiccation
• Stockpiling prior to initiation of control program
• Community engagement
27. Discussion
• What are the advantages/disadvantages of a mosquito control program
featuring genetically modified mosquitoes, such as the one described
here and one being implemented in the Florida Keys by Oxitec?
29. Supplemental Information
• Virus protein 16 (VP16) is a transcription factor encoded by the UL48
gene of Herpes simplex virus-1 (HSV-1)
• The transactivation domain (TAD) of VP16 can be fused to a DNA-binding
domain (DBD) of another protein in order to gain expression of a desired
target gene
Editor's Notes
Due to Aedes aegypti ability to breed in a wide variety of containers, and finding and treating sufficient numbers of them is extremely challenging or impossible for even the most well-funded and organized programs
-Method of biological insect control, whereby overwhelming numbers of sterile insects are released into the wild
-used to successfully eradicate the screw-worm fly from North and Central America
-Previous mathematical modeling of this system predicts that fewer male mosquitoes of a late-lethal strain need to be released as compared to those carrying an early-lethal gene or irradiated strain to achieve an equivalent level of control of a target-population
-Previously constructed repressible lethal strains of insects using the “tet-off” gene expression system based on a synthetic tetracycline-repressible transactivator (tTA)
Biological basis for this differential splicing is unknown
AeAct4-mediated expression of tTA in these dual transgene hybrid mosquitoes produces the tTA transactivator that then binds the operator (tetO) and triggers expression of the reporter gene product (DsRed in this case)
Arrows depict the developing IFMs on the thorax of the larvae
Arrows depict the developing IFMs on the thorax of the larvae
Arrows depict the developing IFMs on the thorax of the larvae
Nipp1Dm = nuclear inhibitor of protein phosphatase 1 (protein phosphatase 1 is important in the control of various cellular processes)
michelob_x = inhibitor of apoptosis protein antagonist (prevents apoptosis inhibition)
-Although expression of tTA at moderate levels appears to be innocuous, expression at high levels can be deleterious, disrupting transcription and the ubiquitin-dependent protein degradation system
-Inferred that the AeAct-4 fragment used represents a strong promoter that expresses tTA at a level close to the threshold for IFM damage (and that expression is modulated by position effects so that different lines show the flightless phenotype at a greater or lesser extent
-Reasoned that the observed flightless phenotype of insects expressing tTA alone was due to VP16, the domain of tTA that can cause cellular toxicity; additional expression of VP16 should therefore increase penetrance of this phenotype
-Furthermore, this would be combined with the potential advantage of VP16 having low toxicity in basal or off-target expression, and consequent minimal effect on male fitness, or indeed on females reared in the presence of Tc
***Driver component consists of the regulatory region + sex-specific intron of AeAct-4 + tTA with an engineered start codon at the 5’ end of the sex-specific intron
*** Effector component has the VP16 effector under the control of the tTA response element (tRE)
