Dear Nicole,

We have knocked in stable GFPs (or GFP-fusions) into around 10 genes.
Some you can see some you cannot.  Some of the ones you can't really see
on a sensitive microscope you can just about see using flow.  Maybe
someone out there has a new Dicty superfolder GFP, which might help.

We initially used crude measures to select genes for tagging- basically
how quick was a decent Northern exposure.  It subsequently turned out that
the ones that worked well have high counts in RNAseq data, such as Gadi's
published developmental time course, or our own data.  One high expresser
didn't work, for no reason we understand.

So overall, I would expect a destabilised GFP to be worse (certainly based
on comparing ES cells with stable and unstable GFP knocked into the Rex1
gene with the same targeting vector).  I think the autofluorescence of the
cells might dominate the signal.  I wouldn't expect it to work for your
gene, although I really hope it does.

With overexpression vectors, it may work, but be careful making inferences
on regulation as you will be missing shadow enhancers, nuclear and genomic
context etc. etc.

Good luck,

Jonathan






On 19/11/2014 14:47, "Nicole Gruenheit"
<[log in to unmask]> wrote:

>Hi all,
>
>thanks for the incredibly fast reply. We were also wondering if there is
>a minimum expression level when using these reporters? Do you need
>something as strong as ecmA/ecmB or can you use a gene with relatively
>low expression level? In my RNA-seq data the gene we are interested in
>has a normalised read count of about 40 while ecmA is 65x higher at the
>12h timepoint.
>
>Cheers,
>Nicole
>
>Dr. Nicole Gruenheit
>
>Research Associate
>Faculty of Life Sciences
>Michael Smith Building
>Oxford Road, Manchester, M13 9PT
>The University of Manchester
>http://thethompsonlab.wordpress.com/
>
>On 18 Nov 2014, at 14:49, Richard Gomer <[log in to unmask]> wrote:
>
>> Yes, Harry MacWilliams did this
>> 
>> Dev Genes Evol. 1999 Jan;209(1):63-8.
>> Green fluorescent proteins with short half-lives as reporters in
>>Dictyostelium discoideum.
>> Deichsel H1, Friedel S, Detterbeck A, Coyne C, Hamker U, MacWilliams HK.
>> 
>> We describe two modifications of the popular reporter green fluorescent
>>protein (GFP) which have short half-lives in our system, the cellular
>>slime mould Dictyostelium discoideum. One of these bears an N-terminal
>>ubiquitin; this GFP was originally planned to be a substrate of the
>>"N-end-rule" pathway, but deubiquitination does not seem to occur, and a
>>degradation by the UFD (ubiquitin-fusion-degradation pathway seems more
>>probable. The protein half-life is about 3-5 h. The second construct has
>>an N-terminus derived from the L11 ribosomal protein; it is transported
>>to the nucleus and broken down much more rapidly than the ubiquitin
>>fusion (protein half-life about 30 min). We show examples of the use of
>>these reporters in the study of gene expression in Dictyostelium.
>> PMID: 9914420 
>> 
>> cheers
>> 
>> Richard Gomer
>> 
>> 
>> ________________________________________
>> From: DICTY [[log in to unmask]] on behalf of Nicole
>>Gruenheit [[log in to unmask]]
>> Sent: Tuesday, November 18, 2014 8:12 AM
>> To: [log in to unmask]
>> Subject: [DICTY] destruction/degeneration sequence motifs/signals in
>>Dicty
>> 
>> Does anybody have experience with destruction/degeneration sequence
>>motifs/signals in Dicty?  I want to make "labile" promoter::reporter
>>gene (e.g. GFP/RFP) constructs where the GFP/RFP protein is rapidly
>>degraded (e.g. by ubiquitin/proteosome pathway).  Thank you for your
>>help, Nicole and Thomas