Dear colleagues,
Thank you for all of your suggestions and interesting ideas.
I forgot to specify that we'd like to observe the native promoter activity in quantitative way (for our future screening plan), so that we shouldn't use multiple-copy transformants.
I didn't know about IRE, and it's attractive because it may be the easiest one to test whether it will work in Dicty or not.
I also got suggestion that VP16 should work in Dicty as it works in yeast and plant as well. So we might be trying Gal4-VP16 method.
IRES might be the most risky one as it would take years to explore enhancer element sequences. It might simply doesn't exist in Dicty.
Many Thanks,
Koki
Koki Nagayama, PhD
Research associate
Faculty of Life Sciences,
University of Manchester,
Michael Smith Building,
Oxford Road,
Manchester
UK
M13 9PT
________________________________________
From: Michael Myre [[log in to unmask]]
Sent: Wednesday, April 25, 2012 5:31 PM
To: Koki Nagayama
Subject: Re: [DICTY - 779] Signal amplification of weak expressions
Have you explored intron-mediated enhancement (IRE) which is similar to
IRES? It works well in many systems using either GFP or a luciferase
reporter. http://www.nature.com/gt/journal/v8/n8/full/3301440a.html
I am not sure if anyone has explored enhancer elements in Dicty though, so
IRES may or may not work. With IRE, rather than having your promoter of
interest drive expression of a cDNA, you incorporate into the cDNA a false
intron and a polyA site at the end of the transcript. This can increase
reporter expression over 40 fold. Beetle luc is also brighter than
firefly...that too might be an option.
How does the gene expression look like by qRT compared to the rnlA gene?
Another option is using a vector that allows for expression of two genes
in opposite orientations under control of your promoter. The two genes can
be enhanced luciferase. I would IRE first.
Good luck.
Mike
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]
>
----------------------------------------
Michael A. Myre, PhD
Center for Human Genetic Research
Massachusetts General Hospital
Harvard Medical School
Richard B. Simches Research Center
185 Cambridge St., CPZN 5.612A
Boston, MA 02114
Ph. 617-643-5536
Fax 617-726-5735
[log in to unmask]
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