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To design a experiment in feeding of C. elegans. It has to choose a plasmid vector to insert the gene of interest that can feed to C. elegans. Many paper are using pL4440 for the feeding vector, saying it is the first used in feeding C. elegan and because of the two T7 promotor, so it easy to perform the experiment. There also paper saying T3 promotor is not fit for feeding in C. elegans. I am curious about why T3 is not fit? and because I didn't see many paper using other vector to perform the feeding experiment in C. elegan. So I wonder that is it ok if I choose a plasmid that has two T7 promotor and is used to express in worms like pLT61, will it influence the feeding or the efficiency so that people use pL4440 for feeding in C.elegan? Or is people use pL4440 vector because it is specific designed to the feeding experiment of C. elegan? Thank you!

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PREFACE: There's a text, C. elegans: Methods and Applications, specifically doi: 10.1385/1-59745-151-7:109. They outline this exact experiment in it's essence, and one of the diagrams (page 110) notes that for the purposes of RNAi in C. elegans, the T3 promoter specifically doesn't work for the bacterial feeding method, but works for injection or soaking procedures. I absolutely can't figure out why that is specifically, but i'll keep researching.

UPDATED: Much thanks to mdperry for clearing things up! As it turns out, this is a matter of experimental design. Using information from Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans, and Source Bioscience:

HT115 (DE3) is a feeding strain engineered like so,

The genotype is as follows: F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNase III minus). This strain grows on LB or 2xYT plates (and is resistant to tetracycline, see below), and competent cells can be made using standard techniques.

We can go through these and look at the changes as well:

F- means no F plasmid for conjugation. mcrA/B refers to mutations made which reduce the ability of the E. coli to attack foreign DNA. IN(rrnD-rrnE)1 is an inversion in the region of some rRNA operons that I'm unsure as to the effect. Lambda- removes the lambda lysogen. You can see that they have the lavUV5 promoter for an IPTG-inducible T7 polymerase which is important for getting the E. coli to begin producing T7 polymerase to express the associated cloning vector. You can also see that HT115 (DE3) is deficient for RNase III, however, which normally degrades dsRNA, making it suitable for the feeding experiment.

The L4440 (pPD129.36) cloning vector contains two convergent T7 polymerase promoters in opposite orientation separated by a multicloning site and was made using standard cloning techniques (Timmons and Fire, 1998).

pLT61 contains 0.8 kb of unc-22 inserted into the L4440 vector.

I think the L4440 plasmid is probably easy to optimize for this experiment, and cheap. If you can access the article I linked above, they catalogue some additional plasmids. In conclusion though, it'd seem that if we in fact had a system where the E. coli themselves could produce T3 polymerase, it'd be fine to use those promoters. In the case of this experiment, the genotype of the feeding strain of bacteria nicely complements the vector being used.

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    $\begingroup$ One also needs to use the appropriate bacterial host. As I recall HT115 is engineered to carry a copy of the gene for bacteriophage T7 RNA Polymerase, and this polymerase is being driven by the lacUV5 promoter. So in the absence of an inducer for the lac operon the T7 RNAPol is not made, and the dsRNA is not made. When you add IPTG the polymerase is turned on, and the insert in pL4440 is transcribed from both strands. The reason you can use the T3 promoter for injections or soaking is because the T3 RNAPol is added to the test tube. $\endgroup$ – mdperry Oct 17 '15 at 20:24
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    $\begingroup$ Is there a molecular explanation for why T3 isn't fit for feeding ,i.e. the same inducible system doesn't work for T3 polymerase? Or is it just a matter of experimental design? $\endgroup$ – CKM Oct 18 '15 at 17:59
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    $\begingroup$ This article shows the effect of a stem-loop GFP-unc-22 structured dsRNA in RNAi. Basically, it can potentially knock down both GFP and unc-22, and what this does to the worms is induce a twitching phenotype because it alters the contractility of the muscles. It also produces issues with egg laying. $\endgroup$ – CKM Oct 26 '15 at 19:13
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    $\begingroup$ If your endgame is to simply knock down GFP expression and visualize that and be done, there may not be a huge effect, but prolonged exposure to unc-22 knockdown might be deleterious to the numbers of worms in your culture. Let me know if anyone more experienced has input! $\endgroup$ – CKM Oct 26 '15 at 19:13
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    $\begingroup$ In that article, though, they did find that unc-22 dsRNA exposure had to be rather continuous to evoke unc-22 knockdown, so injection with a specific amount of dsRNA didn't work for long. $\endgroup$ – CKM Oct 26 '15 at 19:18

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