Dear Koki,
I know nothing of the two approaches you've mentioned but, depending 
upon the selective agent used to introduce your reporter construct, 
there may be the chance of boosting expression simply by raising the 
drug concentration.  In our hands boosting G418 from 15 to 30, 60, or 
even 120 ug/ml did increase (several fold, no more) GFP expression 
driven by either the actin or PSA promoters.  An increase in integrated 
array copy number might be the tempting explanation, but our Southern 
blots at the time suggested instead some other explanation, ie "epigenetic."
David

On 4/23/2012 1:46 PM, Koki Nagayama wrote:
> Dear Colleagues,
>
> Does anyone know any method to amplify reporter gene expression level in order to visualize weak promoter activities in vivo in Dicty?
>
> For example in mammalian, there are several methods such as IRES and TSTA:
>
> When an internal ribosomal entry site (IRES) sequence is placed upstream of a reporter gene (at the second cistron position), it greatly increases translation rate per mRNA. Thus, a signal from a weak promoter will be amplified translationally by 10 -100 folds.  This system works fine in mammalian, but does anyone know if this would also work in Dicty? IRES sequence might be different in Dicty?
>
> The other method TSTA, two-step transcriptional amlification,  amplifies the transcription level. Your promoter is used to express GAL4-VP16, which is a strong activator in mammalian and the reporter gene will be transcribed by 10 - 50 folds. However, in order to try this method in Dicty, I think a strong activator that works in Dicty needs to be used instead of VP16.  Has anybody tried this kind of method? or Is there a very strong activator (or very strong repressor) in Dicty which might work in this system?
>
> Or is there any other method to make invisibly weak expression visible?
> Any suggestion or comment will be appreciated, too.
>
> Many thanks in advance,
> Koki
>
> Koki Nagayama, PhD (Chris Thomson lab)
> Research associate
> Faculty of Life Sciences,
> University of Manchester,
> Michael Smith Building,
> Oxford Road,
> Manchester
> UK
> M13 9PT
>
> [log in to unmask]

-- 
David I. Ratner
Department of Biology
Amherst College, Amherst MA, USA 01002-5000

phone: 413-542-2248
fax:   413-542-7955
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