Can anyone advise on suitable Gateway-adapted 35S vectors for transient expression in Arabidopsis?
It is worth visiting the University of Ghent website (http://gateway.psb.ugent.be/) and explore their GATEWAY vector resource. If you are an academic researcher, you can establish an MTA and order their vector series (its not expensive). we have used various vectors from them and they work fine in transient expression assays in N. benthamiana.
It is worth visiting the University of Ghent website (http://gateway.psb.ugent.be/) and explore their GATEWAY vector resource. If you are an academic researcher, you can establish an MTA and order their vector series (its not expensive). we have used various vectors from them and they work fine in transient expression assays in N. benthamiana.
We even published a transient expression protocol to use for Arabidopsis roots (Van Loock et al., 2010, plant Signaling & Behavior) if you are interested.
I second Edgar in recommending these vectors from Ghent, we've used them with good success too. But take a closer look at the composition of the vectors you are choosing from that source. Most of the ones we used contain a 2x35S* promoter giving huge amounts of expressed protein. This, especially if looking at subcellular localisation, may lead to artifacts probably caused by misfolding. If this will be a problem for you too, I'd recommend the pGWB series from Nakagawa (http://shimane-u.org/nakagawa/gbv.htm). The only thing you have to consider there is the amount of Hygromycin B you put into your media (up to 150µg/ml). Do you mean Agrobacterium-mediated by transient expression in Arabidopsis? If so, you should include "empty vector" and "empty strain" controls because most of our strains give terrible background reactions on Arabidopsis (but not on tomato, pepper, potato and a lot of other nightshades). HTH, Robert
If you want to obtain the information for Gateway vector in more detail, contact Tsuyoshi Nakagawa ([email protected]).
You can use PMDC-83, 84 85 or PMDC-43, 44, and 45 for either N-terminal or C-Terminal fusion protein. These are Mark Curtis Gateway vectors.
Here is the link-
http://botserv1.uzh.ch/home/grossnik/curtisvector/index_2.html
Thanks
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