Perform loss-of-function studies of your gene of interest
A common way to test what proteins do in biological processes is to perform loss-of-function assays. Phenotypic readouts of loss-function cells/organisms give indications of what a specific protein function does.
Fusing GFP (or another fluorophore) to the gene of interest (GOI) enables visualization of the cells and sublocalization of the GOI. However, without modulation of protein expression one cannot know what the function of the protein is. Fluorescent protein RNAi bacteria can be used to knock down any protein that is fused to GFP.
What is NemaMetrix’s Fluorescent Protein RNAi Bacteria?
- It’s designed to inducibly express interfering RNA (RNAi) that targets GFP mRNA for degradation
- It can be fed to C. elegans as their food source so they are exposed to the RNAi as they eat and grow
- It will knockdown GFP and any protein to which it is fused by feeding the bacteria to worms
Fig. 1. In CRISPR GFP insertion lines, progeny of L4/adult worms exposed to induced GFP RNAi bacteria lose expression of their gene of interest (GOI) that is fused to GFP. L4/Adult GFP inserted worms were plated on either control (HB101) or GFP RNAi bacteria. After 48hrs, eggs were imaged for GFP. GFP RNAi treated eggs do not express GFP, while control eggs do.
- Reduce expression of anything GFP tagged instead of individual RNAi bacterial strains for each gene.
- Use the exact same strain and genetic background as an experimental control for functional studies. Better controls!
- Knockdown your endogenous protein of interest
- Provides visual feedback on the level of inhibition of the GOI
You can now:
- Perform loss-of-function analysis of your gene of interest in the same strain as your other experiments
- Test on different GFP expressing strains with the same bacteria plates
- Induce phenotypes as seen in gene deletion strains
Our GFP RNAi is:
- Specific to NemaMetrix built GFP strains – other targeting constructs do not inhibit our GFP expression.
- Shown to effectively knockdown expression of GFP and the fused Gene of Interest
Figure 2 (Right). L4/Adult GFP-inserted worms were plated on either control (HB101) or GFP RNAi bacteria. After 4 days, an image of the bacteria seed was imaged. In this example, GFP is fused to hrp-1. RNAi against hrp-1 is lethal. This indicates that depleting a GFP fused form is also lethal.