In article <841530807C0 at csnet.nw.uoguelph.ca>, ACARLSON at CROP.UOGUELPH.CA
("Alvar Carlson") wrote:
> On April 14th I posted the following questions:
> > I am attempting to use sGFP to visually select transgenic barley.
> >I am concerned that the accumulation of the GFP in the nucleus will
> >be detrimental to the development of transgenic plants. Has anyone
> >observed reduced transformation efficiency in plants when using GFP
> >or reduced fertility in any of the transgenic plants generated?
> >Furthermore, has anyone found that modified GFP, to exclude it from
> >the nucleus, increases the transformation efficiency?
>>> From: David Galbraith <dgalbrai at ag.Arizona.EDU>
>> We have expressed GFP transgenically targeted to the nucleus in tobacco and
> have seen no evidence of toxicity. Plants have not gone to the next
> generation yet.
>> From: sjdavis1 at students.wisc.edu (Seth J. Davis)
>> WT GFP is toxic, and interferes with transformation efficiency. Modified
> GFPs (smGFP or mGFP5) appear to be less toxic in regenerating plants.
> mGFP5, which is ER localized (not in the nucleus) might reduce the toxic
> effects of GFP. This last point is not clear; is making GFP soluble or is
> localizing GFP to the ER increasing transformation efficiency? This has
> not been tested.
>> From: krs1 at mrc-lmb.cam.ac.uk (Kirby Siemering)
>> We found it necessary to remove GFP from the nucleus by localising it to
> the ER in order to successfully regenerate bright and healthy Arabidopsis
> plants. See Haseloff et al PNAS 1997 (94) 2122-2127 and
>http://brindabella.mrc-lmb.cam.ac.uk/> for details.
A couple of comments. Firstly, wt GFP is largely insoluble when expressed
in bacteria at 37 degrees. However, it is >90% soluble when expressed at
25 degrees. The insolubility at higher temperatures is due to a
temperature-dependent lesion in the folding of the apoprotein (Siemering
et al. 1996 Curr Biol 6 1653-1663). These results suggest that wt GFP
should already be soluble when expressed in plant cells at around 23
degrees, although this has not been proven one way or the other.
Secondly, we did not find wt GFP to result in a lower transformation
efficiency per se. In fact, we were able to consistently produce very
bright callus using wt GFP. Our problem was that we were consistently
unable to regenerate this brightest callus into bright and fertile plants.
Localisation of GFP to the ER has appeared to cure this problem with very
bright callus being produced which can successfully be regenerated into
fertile plants that show high levels of fluorescence. See the
above-mentioned PNAS reference for details and the web site for an image
of a mgfp5-ER expressing adult plant under UV lamp illumination.
Dr. Kirby Siemering
Division of Cell Biology
MRC Laboratory of Molecular Biology
Cambridge. CB2 2QH
Ph. (01223) 402024
FAX (01223) 402008
email krs1 at mrc-lmb.cam.ac.uk