Why do OX5034 GM mosquitos require the presence of tetracycline to survive? What does the drug do in this case?

Question

I'm confused. Debug Fresno; why are the released mosquitos said to be sterile? from 2017 addresses male mosquitos released with a bacteria that will affect fertility of females after mating. They are not genetically modified.

CNN's 2021 article First-ever US release of genetically modified mosquitoes begins in Florida Keys describes release of Oxitec's OX5034 GM mosquito Aedes aegypti. Curiously, these can be kept viable for reproduction when maintained in an environment that includes tetracyline, an antibiotic.

Question: Without the bacterial infection, is it just a coincidence that the GM strain needs an antibiotic to survive? What does the tetracycline do in this case? Why was this drug chosen?

Potentially helpful resources:

• https://www.oxitec.com/en/our-technology
• https://endmalaria.org/sites/default/files/Enca%20Martin-Rendon.pdf

Answer 1

They don't go into any detail on the sites you link to and I don't have time to look into patents, but from the context it sounds like they may be using a tetracycline repressible promoter. This is often referred to as a Tet-Off promoter.

The core elements of these promoters were discovered in the bacterium Escherichia coli¹ and several variations have been developed. These types of promoters work in organisms from bacteria to yeast to mammals^1-3.

References

1: Gossen, M., & Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences, 89(12), 5547-5551.

2: Baron, U., & Bujard, H. (2000). Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods in enzymology, 327, 401-421.

3: Orth, P., Schnappinger, D., Hillen, W., Saenger, W., & Hinrichs, W. (2000). Structural basis of gene regulation by the tetracycline inducible Tet repressor–operator system. Nature structural biology, 7(3), 215-219.

Answer 2

The other answers are correct and on the right track, but I will expand on them with the specific mechanism for these mosquitos. The following paper discusses the engineering behind strain OX513A upon which the OX5034 strain is based:

Phuc HK et al. 2007. Late-acting dominant lethal genetic systems and mosquito control. BMC Biol 5:11

Oxitec mosquitos carry a transgenic construct containing two genes. One encodes for a fluorescent marker, DsRed2, for identifying genetically modified mosquitos and the other encodes a protein called tTAV:

The tTAV gene is under the control of a minimal hsp70 promoter flanked immediately upstream by a tetO site. The tTAV protein is a transcriptional activator that binds tetO and enhances expression. What that means in this construct is that expression of the tTAV gene creates more tTAV protein which binds to tetO which leads to more tTAV expression, etc. This is the positive feedback mechanism mentioned in the above image. Importantly, however, the tTAV protein can bind tetracycline and, when complexed, can no longer bind tetO and therefore cannot drive its own expression. In other words, in the presence of tetracycline, there will be minimal tTAV expression.

tTAV overexpression is lethal during development. Although the exact mechanism of lethality isn’t entirely clear, there are some leading hypotheses:

Knudsen KE et al. 2020. Genetic Variation and Potential for Resistance Development to the tTA Overexpression Lethal System in Insects. G3 10(4): 1271–1281

tTA overexpression is thought to cause lethality due to “transcriptional squelching,” that is a general interference in gene expression (Gong et al. 2005). Consistent with this hypothesis, some genes identified by the GWAS were involved in gene silencing (Su(var)2-HP2) (Shaffer et al. 2002; Shaffer et al. 2006), chromatin binding (mamo) (Hira et al. 2013), chromatin remodeling (Hira) (Loppin et al. 2000) and alternative splicing (bru1)(Spletter et al. 2015), which could all influence the level of tTA expression. Other candidate genes were involved in defense response (PGRP-LC, Lmpt)(Jin et al. 2008), the septate junction (cora) (Tepass et al. 2001) and apoptosis (out) (Coffman 2003); all of which are systems that could potentially impact survival. Four genes; eff, tey, CG32085 and CG13085, encode proteins that are predicted to participate in protein ubiquitination and degradation (Thurmond et al. 2018; Gramates et al. 2017). For example, the Eff protein is a E2 ubiquitin-conjugating enzyme (Chen et al. 2009). It has been suggested that overexpression of the tTA protein could cause lethality due to interference with ubiquitin-dependent proteolysis (Gong et al. 2005) as ubiquitination of VP16 is required for activity and also signals destruction (Salghetti et al. 2001).

To summarize, tTAV is a transcriptional activator and, in Oxitec mosquitos, it regulates its own expression in a positive feedback loop: tTAV protein binding to its response element tetO leads to more tTAV gene expression which leads to more tTAV protein. This overexpression of tTAV is lethal during development. However, tTAV can also bind tetracycline and, when it does, it can no longer bind tetO. In the presence of tetracycline, there is no positive feedback loop and and only basal, non-lethal amounts of tTAV are expressed.

Image from the Oxitec website.