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The experiment that is using C. Elegans to silence the Genes. I have a question about Why and how C. Elegans can use the DNA plasmid that is generated with the gene of interest in the bacteria by being fed the bacteria?

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  • $\begingroup$ If you describe your procedure in a little more detail it will be easier to tell exactly what you are doing, but basically the cells of the C. Elegans can be induced to take up the plasmid and that can be transcribed by an RNA polymerase. The Messenger RNA is then translated by the ribosomes to produce a protein that inhibits the transcription of the gene of interest. If it is RNAi, then the RNA produced will hybridize with the mRNA transcripts that are being produced by the gene of interest. dsRNA cannot be translated and even if some of the mRNA is, the protein will be nonfunctional. $\endgroup$ – AMR Sep 7 '15 at 18:44
  • $\begingroup$ If you are actually doing recombination, then you likely linearized your plasmid with a restriction enzyme and the plasmid has homology arms that will allow it to be combined into the genome of the worm's cells. The success rate is usually low without some form of technology like CRISPR or Talens, so if you are using those, then there is likely some form of targeted DNA break that is happening to allow for homologous recombination to integrate the linearized plasmid into the chromosome of the worm. $\endgroup$ – AMR Sep 7 '15 at 18:47
  • $\begingroup$ It sounds like you are performing RNAi... this procedure explains what is going on. jove.com/science-education/5105/rnai-in-c-elegans They take the bacteria up in vesicles, digest then which releases the RNAi that the bacteria has produced and that intern goes on to hybridize with the mRNA of the gene of interest, as I said above, that binding either stops translation altogether or the protein product is no functional. $\endgroup$ – AMR Sep 7 '15 at 18:55
  • $\begingroup$ If it's what I'm thinking of, you probably have GFP+ worms that end up getting fed some DH5-α E. coli that were transformed using CaCl2. The outcome is your control worms should nicely fluoresce, and the experimental worms will be a noticeably weaker color. The RNAi process is explained nicely in that link provided by AMR. $\endgroup$ – CKM Sep 7 '15 at 20:16
  • $\begingroup$ Thank you AMR and Kendall for explaining this to me!! and Thanks for the video. It helps a lot to understand the concept. Thank you!!! $\endgroup$ – Scsqpd Sep 7 '15 at 21:12
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From the way that you have described your experiment, feeding C.elegans bacteria that are expressing a plasmid that will induce gene silencing, then both @Kendall and I think that you are likely performing an RNAi (interference) experiment.

As @Kendall writes:

If it's what I'm thinking of, you probably have GFP+ worms that end up getting fed some

DH5-α E. coli that were transformed using CaCl2. The outcome is your control worms should nicely fluoresce, and the experimental worms will be a noticeably weaker color. The RNAi process is explained nicely in that link provided by AMR. – Kendall

The bacteria will transcribe RNA molecules encoded by the DNA plasmid. These RNAs will have complimentary sequences to sequences from your gene of interest.

When the nematode eats the bacteria, they RNA molecules that the bacteria has produced will be taken up by the cells of the worm.

The Dicer enzyme then cleaves the dsRNA into small fragments. These fragments are then able to hybridize with the mRNAs transcribed from the gene of interest. This will either cause the ribosomes not to bind properly or will start translation out of frame or without key amino acids or will terminate translation early.

The net effect is that the cells will either not produce the protein encoded by the gene of interest or will produce a non-functional protein. If, as @Kendall said, the gene of interest is a reporter gene such as GFP, then the result of the interference experiment will be C.elegans that do not fluoresce. If the gene of interest is a structural protein such as actin, then you will see the effects of knocking out the actin gene on the cells of the worm.

This online video protocol from JoVE.com gives a detailed overview of the RNAi in C.elegans experiment and also demonstrates how the protocol is carried out.

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