A question about studying plant gene subcellular localization byusing eYFP

Dear all,

I have tried to study the subcellular localization of my gene by fusing it
in frame with eYFP and also DsRed2. I have placed it under the control of
CaMV and I have sequenced the constructs and I am sure that the construct is
correct and there is no misake in the construct. I tried to overexpress the
construct in transgenic Arabidopsis and since my construct also contains
CaMV GUS I found that my transgenics are GUS positive but I can't see the
fluorescence of eYFP/ DsRed2. Is it possible that the fluorescent proteins
doesn't fold correctly when it is fused with my protein of interest and so I
can see the fluorescence? does anyone have similar experience to share? What
can I do now?


Thanks in advance
Kam Ho

Hi Kam,

had similar problems with my constructs (in pAVA vectors) changing to
another vector with a different linker between GFP and my cDNA worked. Also,
you might try transient expression by particle bombardment as you can
increase the amount of DNA you shoot and thus the expression of your
construct.

Cheers
Oliver

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"Chan Kam Ho" <[Only registered users see links. ]> wrote in message
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is
the
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What

Dear Kam and others,
Oliver's suggestion to use transient expression techniques when
evaluating fluorescent protein expression from your vectors is a good
one. As well as particle bombardment techniques, we use a
transient-expression-in-tobacco-leaves trick that is much simpler and
quicker than stably transforming arabidopsis. This has just been
published as Sparkes et al (2006) Nature Protocols 1: 2019-2025. If
you would like a pdf, let me know.
Cheers, John.
Oliver Berkowitz wrote:

Hi Kam,

had similar problems with my constructs (in pAVA vectors) changing to
another vector with a different linker between GFP and my cDNA worked. Also,
you might try transient expression by particle bombardment as you can
increase the amount of DNA you shoot and thus the expression of your
construct.

Cheers
Oliver



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Oxford Brookes University
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