Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes
July 4th, 2007
Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.
Every 15 seconds, there is a child dying from malaria in the world, in here in the James lab, or trying To make French mosquitoes that cannot carry the malaria persons. My name is TIUs. I'm here at Universal of California Irvine at Department of Molecular Biology and Biochemistry in the lab of Anthony James.
Today I will show you how to make transgenic and not Stevens eye. And we are doing this to make malaria mosquitoes refractory so they cannot Carry the malaria parasite. Now I have prepared a vial with Isotonic buffer where the mosquitoes are going to lay their eggs.
I'm gonna use this aspirator to suck up the mosquitoes and put in a vial. This vial is then going into an incubator that is 30 degrees Celsius, and we'll leave it for about one hour in a cup. Now we're gonna pull the microinjection needles.
These are needles made of quartz and we have to use a very fine tip. Therefore, we need these needle puller. We use a needle puller from Suter instruments and also the needles from subtle in SU instruments.
So now taking out the mosquitoes and I will put these eggs down into the Petri dish where I have a filter paper soaked in isotonic buffer that is just to keep the embryos wet during the injection period. The amount of water on the filter paper is critical. You don't want the eggs to float around and you don't want it to get dry.
Now I'm gonna move the embryo from the egg paper onto another three MM watman paper that has been soaked in isotonic buffer. Now do this to take away the Corian before aligning them up for the microinjection. So now I'm gonna take away the Corian, which is this silvery surface on the eggs.
And I do that by brushing them off with my brush. So on top here we have an embryo where the Corian is still sitting and they have this silvery shining part. Where is the one below?
I have been successful to taking out the Corian and this the top one will probably slip on a tape, whereas the bottom one will stick. And that is what's important when you do an off even side. Now I am aligning the embryos.
The embryos are factor or bigger on the anterior end, and that one I'm placing to the right. On the left I have the poster end, which is slimmer and which I, where I will inject the embryos. I'm trying to put them together so they help each other to stay in position.
And usually I do about 10 in a line together. Now I have a line, the embryos, and I will transfer them to a cover slip. On the cover slip.
I put wig tape in order for them to stick. When I'm done that I will dry the filter paper where the embryos are sitting with another filter paper, put the embryos on the cover slip and let it dry for 30 seconds before I apply oil. Now we have aligned the embryos and have them under the oil.
On the microscope stage, we will inject DNA mixed with food dye using this special tip of the microbe pattern And inject this one into the port's needles. Now we have filled the needle and put it back on a micro injector. And now we're ready To do the micro injections.
Now I'm moving in the needle to sharpen it and I, so I can see it focused. Once that is done, I move a weight needle and I move in the embryos and focus those as well. And when that is done, I'm moving the needle again until I have it in position and I move on my embryos.
Now when the needle gets through the oil, it gets blurry and you need to adjust it again. So that's exactly shark. Exactly, shark tick.
And now I'm ready to do the injections. You could see that I touched the embryo with the needle, and by doing that I broke the tip and you can see a small droplet coming out that would be injected into the embryo. So here inject and get it out again.
And I check every time between each embryo that I have a small droplet coming out to the tip of the needle. And as you can see, I need to readjust for each embryo to get in perfect position. Now this one was leaking.
As you can see, when I do my second injection out in the oil, the bubble becomes very large and I need to readjust the pressure of the micro injector. So now we're done with the micro injections and we're going to move the embryos from the microscope into the Petri dish, which we have filled with isotonic buffer. I will wipe off the oil from the cover slip and cut it into a small piece, which I put down into the Petri dish.
Now we're gonna leave them here until the end of the day and room temperature. And then we move Them down to into the insectory. Today I've shown you how to inject an awfully Steven side To make transient mosquitoes.
And now we just have to wait for the larvae to hatch all the larva to hatch. We will bring up to adults and then they will mate. And after that we're gonna have three weeks before we can start a screen if they're Refactor or not.
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Overview
This video demonstrates the technique for performing microinjections in Anopheles stephensi mosquitoes, which are vectors for malaria in Asia. The methodology shown is applicable to other mosquito species as well.
Key Study Components
Area of Science
- Neuroscience
- Genetics
- Vector Biology
Background
- Anopheles stephensi mosquitoes are significant malaria vectors.
- Malaria poses a severe health threat, particularly to children.
- Transgenic approaches aim to create mosquitoes that cannot carry malaria.
- Microinjection techniques are crucial for genetic modifications.
Purpose of Study
- To develop transgenic Anopheles stephensi mosquitoes.
- To confer malaria resistance to these mosquitoes.
- To demonstrate microinjection techniques applicable to various mosquito species.
Methods Used
- Preparation of isotonic buffer for mosquito egg laying.
- Microinjection of transgenes into mosquito embryos.
- Observation of transgenic traits in developed mosquitoes.
- Application of techniques to enhance genetic resistance to malaria.
Main Results
- Successful microinjection of transgenes into Anopheles stephensi.
- Development of mosquitoes exhibiting resistance to malaria.
- Demonstration of the applicability of techniques to other species.
- Potential implications for malaria control strategies.
Conclusions
- Microinjection is a viable method for creating transgenic mosquitoes.
- Transgenic Anopheles stephensi can be engineered for malaria resistance.
- These techniques may contribute to reducing malaria transmission.
Frequently Asked Questions
Executive Industry Relevance
Genetic modification of disease vectors offers a preventive strategy for infectious disease control by targeting transmission at the source. Microinjection techniques enable precise germline transformation in mosquitoes, supporting target validation for antiparasitic interventions. This approach contributes to mechanistic de-risking in early-stage vector control programs by providing a scalable method to assess gene drive and resistance mechanisms.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables functional interrogation of antiparasitic genes in vivo to validate targets for transmission-blocking strategies.
- Operational Value: Provides a reproducible method to generate transgenic lines for phenotypic screening of malaria resistance traits.
Screening & Assay Development
- Scientific Value: Generates standardized mosquito embryos for quantitative assessment of transgene expression and parasite inhibition.
- Operational Value: Establishes a platform for high-throughput screening of genetic constructs affecting vector competence.
Translational & Preclinical Research
- Scientific Value: Supports evaluation of disease-relevant systems by linking genetic modifications to reduced Plasmodium falciparum development in mosquitoes.
- Operational Value: Enables continuity from gene discovery to preclinical validation through stable transgenic mosquito lines.
Pipeline & Workflow Integration
The microinjection workflow fits within the discovery-to-preclinical continuum by enabling target validation, assay-ready model generation, and phenotypic readouts for transmission-blocking candidates.
- Discovery Biology: Facilitates hypothesis testing of antiparasitic mechanisms through embryo microinjection and trait analysis.
- Screening: Produces standardized embryos suitable for assay development and compound or genetic construct evaluation.
- Analytics: Enables quantitative measurement of parasite load and survival rates as dependent variables in resistance screening.
- Translational Research: Connects genetic modifications to preclinical outcomes via adult mosquito phenotype assessment and mating studies.
- Enterprise Reuse: Establishes a reusable platform for iterative testing of genetic interventions across vector species.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through direct phenotypic observation of malaria resistance in transgenic mosquitoes.
- Operational Value: Standardized embryo preparation and injection protocols enhance reproducibility across laboratories.
- Strategic Value: Informs go/no-go decisions by reducing biological uncertainty in transmission-blocking approaches.
- Portfolio Impact: Enables risk-adjusted prioritization of genetic constructs based on demonstrated resistance phenotypes.
Implementation Considerations
- Requires expertise in mosquito embryology and microinjection instrumentation.
- Dependence on quartz needle preparation and precise pressure control during injection.
- Necessitates isotonic buffer maintenance and environmental controls for embryo viability.
- Involves cross-team coordination between entomology, molecular biology, and imaging groups.
- Limited by embryo availability and injection efficiency, which affect throughput and scalability.
