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random musings of a molecular biologist turned code jockey in the era of big data and open science.

Elements of transgenic disease-vector control (Part 2: altering the mosquitoes)

Part one of this series can be found here

Attention

I am serious when I say that I want your criticisms, suggestions, comments, reference suggestions, etc. Please do visit the comments section and help me out! The most helpful commenters will be acknowledged in the final dissertation document.

The four fundamentals of transgenic vector control:

  1. the ability to introduce transgenes into the mosquito’s genome in a heritable way
  2. discovery or construction effector genes
  3. the ability to control when and where the effector genes are expressed
  4. the ability of your transgenic mosquitoes to have swift, dramatic, and clinically meaningful effects on the native vector population (gene drive)

Introducing Inheritable Transgenes into the Mosquito Genome

Before one can employ transgenic methods for vector control, one must of course have the ability to make transgenic vectors. For mosquitoes, this is quite a bit more labor intensive than for other fly species like Drosophila melanogaster. However, it has been accomplished, and protocols to reliably introduce transgenes into many mosquito species now exist. Unfortunately, the relative difficulty is still high, and it takes roughly 2-3 months before a stable line is established. So while this step can be filed under “accomplished”, work is constantly being done to improve its ease and efficiency.

The method of gene insertion into the genome generally relies on re-purposing the activities of small, “parasitic”, mobile DNA elements called transposons or transposable elements. These elements have the ability to cut themselves out of one part of the DNA and re-insert themselves into another. We can engineer them in the lab to carry our genes instead of the genes that they usually contain. This allows our genes to be inserted into the genome instead of the contents of the original transposon.

Until recently, most if not all transposons used inserted the genes into more-or-less random regions, and in single, double or multiple copies and/or locations [Adelman2004],[Sethuraman2007]. This makes characterizing the effects of the effector genes complicated. However, with the successful implementation of the phic31 site specific integration system in Aedes aegypti and Anopheles stephensi [Thorpe1998],[Nimmo2006],[Isaacs2012], we can now reliably target specific, known regions of the genome for transgene integration of a single copy.

The “heritable” part is achieved through which cells take up the transgene. Only certain types of cells have the ability to project their genetic code into the next generation. In order to have a transgene be heritable by the following generations we must make sure that at least some of the cells that get the transgene are germ line cells (sperm or egg cells).

A peculiarity about fly development is that as the embryo is developing the cells that will become the germ line always position themselves at one end (or pole) of the embryo. They earned the moniker “pole cells”. This makes targeting these cells for the transgene feasible. When the solution containing the transgene construct is injected into the embryos, it is done where the pole cells are. This means that the first set of mosquitoes that might be fully transgenic is not the set that you injected but the first generation of offspring that your injected mosquitoes produce.

Effector Genes

With the ability to integrate transgene constructs into the vectors’ genomes, we need constructs that carry genes that will effect the response that we desire in the vector. Two broad strategies are commonly applied when designing an anti-transmission phenotype.

Vector Population Reduction:

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The only “good” mosquito is a...

The goal of vector population reduction is the same as the conventional vector control modalities. Reduce the size of the vector population to decrease the probability of infectious interactions between vectors and humans.

Conventional tactics include removal of breeding sites through draining of swamps, removal of items that store water (tires, buckets, etc) where larvae develop from populated areas, and of course, chemical insecticidal campaigns. Another, more recent tactic is sterile insect technique (see the relevant topic from part one of this series).

A transgenic tactic for the population reduction strategy is illustrated by Oxitec’s Aedes aegypti strain OX3604C developed with support from the Bill and Melinda Gates Foundation’s Grand Challenges for Global Health Initiative. OX3604C, represents a female-specific RIDL approach which uses a poison transgene that kills any female adult expressing the transgene unless the female is being fed the “antidote” through its water supply.

Releasing enough of these mosquitoes will affect the local mosquito species population in a way that is analogous to SIT.

Vector Population Replacement (Conversion):

Note

The more standard term is vector population replacement. However, I prefer vector population conversion because it is more accurate. We will not necessarily even want to replace the local population with purely lab-produced mosquitoes. What will most likely be happening is that the population goes from having no frequency of our designed trait, to having a high frequency of our trait, but that the rest of the genetic information in the population is still derived from the genomes present in the original population.

It would be as if a brown eyed family moved into a valley where people only had blue eyes. Since the brown eyes trait is usually dominant over the blue eyes trait, the brown eyed phenotype will spread through the valley as generations interbreed. However, it would not be accurate to say that the original brown eyed family replaced the native population.

Vector population conversion is a novel strategy for vector control that only exists within the context of genetically altering the vectors. This strategy actually promises to be the most long-lasting vector-focused intervention; because unlike all vector population reduction tactics, there is a theoretical point in a conversion intervention (the anti-transmission trait works and is present in local populations at near 100%) when the human interaction can be ceased but the intervention continues to function. For population reduction to approach this result, the vector species must not simply be eliminated from the local area, but approach elimination on a continental scale, or more realistically achieve global eradication.

The reason is that these mosquitoes (especially Aedes aegypti) can and do travel long distances, as eggs or larvae, in the backs of trucks (between villages) or in the pools of water collected in super-tankers (transcontinentally). So a local village is only free of the vectors until more migrate into the area. But in a conversion scenario, those migrants mate with the local vector population, and their offspring are assimilated into transmission-deficient mosquitoes. The protection of the local village can be preserved even if some surrounding villages fail to maintain control of their mosquitoes.

An example of a transgenic tactic for population conversion of Aedes aegypti into a transmission-deficient phenotype involves an effector gene that codes for double stranded portion of the target dengue virus as RNA [Franz2006],[Mathur2010]. Because most animal cells have a system that detects and degrades double stranded RNA[1] in a sequence specific manner, this primes the mosquito cells’ antiviral response to specifically attack the dengue virus if the effector gene is expressed in the cell before it gets infected.

Controlling When and Where the Effector Genes are Expressed

Crash Course in Gene Expression:

The following is a very brief introduction to how the expression of genes are usually controlled. Pay specific attention to the parts that mention transcription factors and enhancer regions.

If that was a little over your head, watch this version first, then go back and watch the advanced on again.

Even armed with an effector gene that clears 100% of the pathogen 100% of the time, you will not be successful in limiting transmission if it is not turned on in the right time and place. If your effector gene works best when the pathogen is in the midgut, but your gene is only expressed in the antennae, you have wasted your time.

The region of DNA directly before the sequence of the gene is usually the most influential determinant of the pattern of expression and is referred to as the promoter. It determines when the gene will be turned on (let’s say directly following the ingestion of a bloodmeal), and in which tissue type (let’s say the midgut). The way that this is accomplished is due to the binding of special proteins called transcription factors that recognize specific DNA sequences. Once bound to the promoter they recruit the special machinery needed for the gene to be turned on.

If we wanted to control a transgene in a specific way (turned on after a bloodmeal in the midgut), one way to go about it would be to identify genes in the mosquito that already have a similar expression pattern to the ideal that you want. Then we could copy the promoter from that gene and paste it in front of our transgene[2]. Because we will have replicated the specific transcription factor binding sites (TFBS) that control the original gene, our transgene should inherit a very similar expression pattern.

This is a very common process used to engineer the expression patterns of real transgenes in mosquitoes. In [Moreira2000], the promoter sequences of a gene called carboxypeptidase (normally expressed in the midgut after a meal to help digest it) from Aedes aegypti and Anopheles gambiae were pasted in front of a transgene that causes the cells that express the gene to light up. This type of transgene is called a reporter gene because it allows the researchers to visualize the activity of the promoter used to drive its expression.

From the abstract of [Moreira2000]:

Six independent transgenic lines were obtained with the AeCP construct and one with the AgCP construct. Luciferase mRNA and protein were abundantly expressed in the guts of transgenic mosquitoes in four of the six AeCP lines and in the AgCP line. Expression of the reporter gene was gut-specific and reached peak levels at about 24 h post-blood ingestion.

Achieving Swift, Dramatic, and Clinically Meaningful Effects on the Native Vector Population

In many ways, this is the most difficult part of the puzzle. In order for the transgene to have its self-sustaining properties as well as achieve effective anti-transmission results, it must spread through the native mosquito population to the point that the percent of individuals possessing the gene approaches 100%.

Optimistically assuming that the transgene carries a negligible fitness cost[3], or even a slight fitness advantage, achieving near 100% conversion could take decades. Funding terms for efforts like this in poor nations can be closer to 5 years or less. To enable the population conversion strategies to work, we must come up with genetic “tricks” that cause the gene to spread through a mosquito population much faster than could happen naturally. Efforts to discover or design this gene drive system are an on going and active area in this field.

See also

The topic of gene drive deserves its own post, so I will use this space to link to any future post that tackles the subject in substantial depth.


Footnotes:

[1]Double stranded RNA generally signals that a virus is active in the cell.
[2]The story is of course more complicated than this, and the replication of the original expression pattern may not always be perfect with this simplified method; however, it is suitably explanatory for our purposes at the moment.
[3]By fitness cost/advantage here, I mean that the transgene causes the mosquitoes that inherit it to be either less or more successful at producing offspring, respectively.

Citations:

[Adelman2004]Adelman, Z. N., Jasinskiene, N., Vally, K. J. M., Peek, C., Travanty, E. A., Olson, K. E., Brown, S. E., et al. (2004). Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti. Transgenic research, 13(5), 411–25. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15587266
[Sethuraman2007]Sethuraman, N., Fraser, M. J., Eggleston, P., & O’Brochta, D. A. (2007). Post-integration stability of piggyBac in Aedes aegypti. Insect biochemistry and molecular biology, 37(9), 941–51. doi:10.1016/j.ibmb.2007.05.004
[Thorpe1998]Thorpe, H. M., & Smith, M. C. (1998). In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proceedings of the National Academy of Sciences of the United States of America, 95(10), 5505–10. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=20407&tool=pmcentrez&rendertype=abstract
[Nimmo2006]Nimmo, D. D., Alphey, L., Meredith, J. M., & Eggleston, P. (2006). High efficiency site-specific genetic engineering of the mosquito genome. Insect molecular biology, 15(2), 129–36. doi:10.1111/j.1365-2583.2006.00615.x
[Isaacs2012]Isaacs, A. T., Jasinskiene, N., Tretiakov, M., Thiery, I., Zettor, A., Bourgouin, C., & James, A. A. (2012). Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development. Proceedings of the National Academy of Sciences of the United States of America, 109(28), E1922–30. doi:10.1073/pnas.1207738109
[Franz2006]Franz, A. W. E., Sanchez-Vargas, I., Adelman, Z. N., Blair, C. D., Beaty, B. J., James, A. A., & Olson, K. E. (2006). Engineering RNA interference-based resistance to dengue virus type 2 in genetically modified Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America, 103(11), 4198–203. doi:10.1073/pnas.0600479103
[Mathur2010]Mathur, G., Sanchez-Vargas, I., Alvarez, D., Olson, K. E., Marinotti, O., & James, a a. (2010). Transgene-mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti. Insect molecular biology, 1. doi:10.1111/j.1365-2583.2010.01032.x
[Moreira2000](1, 2) Moreira, L. a, Edwards, M. J., Adhami, F., Jasinskiene, N., James, a a, & Jacobs-Lorena, M. (2000). Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes. Proceedings of the National Academy of Sciences of the United States of America, 97(20), 10895–8. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=27120&tool=pmcentrez&rendertype=abstract