On Jan 2, 2007, at 8:09 PM, Luoping Yan wrote:
>>>> As the staining patterns of my promoter fusion lines are not
>> consistent at al, I want to use another vector pBI121 or pCGN1547.
>> Does anyone know where to get pCGN1547?
>> How do you deal with those lines from 35S containing vector?
>> CAMBIA suggests doing co-transformation strategy to complete
>> remove the influence of CaMV35S, i.e. one vector without
>> selectable marker gene in T-DNA and the other contains only
>> selectable marker gene in T-DNA. But it will involve a lot of
>> work and screening workload would be large.
>> Has anyone tried this and how about the success rate?
I haven't tried the co-transformation approach (yet) but I have
looked into it. Most of the literature I've seen on this indicates
that co-transformation rates are pretty high-- I seem to recall
seeing numbers on the order of 35-75% given. So, this does not seem
like it would be so very much work, and screening for the presence of
your gus reporter gene would not be that hard by PCR.
Still, you do see lots of literature with researchers using GUS
reporters in a standard T-DNA with a linked selectable markers driven
by a strong promoter. I suspect this works well enough in lots of
cases, but probably depends on a lot of things (orientation, nature
of both promoters, etc). Probably trying to separate the selectable
marker promoter and the promoter of interest would help somewhat. I
suspect if the reporter data observed in such experiments is
consistent with data already in hand (e.g. northern data, phenotypic
data), a research probably wouldn't worry about the effects of the
linked marker promoter.
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)