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Experimental RNAi

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...

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RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

RNAi-mediated Double Gene Knockdown and Gustatory Perception Measurement in Honey Bees Apis mellifera

Published on: July 25, 2013

Honey bee functional genomics using symbiont-mediated RNAi.

Patrick J Lariviere^1,2, Sean P Leonard^1,2, Richard D Horak²

¹Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.

Nature Protocols

|December 2, 2022

Summary

Researchers engineered a bee gut bacterium to create a sustained RNA interference response, enabling efficient gene knockdown in honey bees. This novel method, Functional Genomics Using Engineered Symbionts (FUGUES), offers a scalable and less invasive approach for bee functional genomics studies.

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Area of Science:

Entomology
Microbiology
Genomics

Background:

Honey bees are crucial pollinators and model organisms, but their social structure complicates genetic studies.
Current methods like dsRNA injection or feeding for gene knockdown are laborious, harmful, or difficult to scale.
Existing RNAi methods require repeated administration for sustained gene silencing.

Purpose of the Study:

To develop a novel, scalable, and sustained method for gene knockdown in honey bees.
To overcome the limitations of existing RNAi techniques in functional genomics research.
To engineer a microbial symbiont for inducing targeted gene expression reduction in vivo.

Main Methods:

Engineered the bee gut bacterium *Snodgrassella alvi* to express double-stranded RNA (dsRNA).
Utilized Golden Gate assembly for dsRNA plasmid cloning in *Escherichia coli* and subsequent transfer to *S. alvi*.
Colonized adult worker bees with engineered *S. alvi* and verified gene knockdown via qRT-PCR.

Main Results:

Achieved sustained RNA interference response in honey bees via engineered gut bacteria.
Demonstrated significant gene expression reduction (50-75%) throughout the bee body within 5 days of colonization.
Established a 4-week protocol for bee researchers skilled in microbiology and molecular cloning.

Conclusions:

Functional Genomics Using Engineered Symbionts (FUGUES) provides a streamlined and scalable approach for honey bee functional genomics.
This method offers a less invasive and more efficient alternative to traditional dsRNA delivery.
The FUGUES protocol has potential applications for studying other insect and animal species by engineering microbial symbionts.