Great suggestions Koki!  I hope you get some good responses.  I'll ask around at UBC and see if anyone is familiar with the techniques.

Hope your well, ta-ra

Alex


________________________________________
From: DICTY [[log in to unmask]] on behalf of Koki Nagayama [[log in to unmask]]
Sent: 23 April 2012 10:46
To: [log in to unmask]
Subject: [DICTY - 779] Signal amplification of weak expressions

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]