GFP expressing stable cell lines

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sat Mar 30 11:47:33 EST 2002


Dear Mauricio, 

I have not solution to your problem, but am curious: How does the spacer look like you are employing?

Did you consider to add some more protein inbetween (like some natural 
occurring spacers that separate domains of diffrent functionality?

Regards,
Wo

> 
> By the way of the messages from Vemula and Wolfang, here in the lab we are
> dealing with a subcloning involving GFP fused to a coiled coil region of an
> interest protein.  Until now we have not get any sucess and I figure out if the
> coiled coil agregation capabilities could explain the lack of a bacterial clone
> expressing the fluorescence.
> 
> Has anybody get similar results getting fusions of GFP to highly
> hydrophobic regions ?.  Any comment would be appreciated. TIA.
> 
> Mauricio
> 
> On 30 Mar 2002, Wolfgang Schechinger wrote:
> 
> > Dear Vemula, 
> > 
> > as long as you don't use some *fast* ripening GFP variant, you won't see much
> >  in that short time.
> > 
> > If you still don't see anything glow after say 1 day, you're in trouble,
> > though.
> > 
> > Basically, it could have happened that you selected for cells expressing NeoR
> > but that have lost GFP for some reason.
> > 
> > Maybe you could include some inductor in the medium when selecting the cells
> > and check them for GFP fluorescence, if your promoter is not somewhat leaky.
> > Weak GFP fluorescence sometimes is difficult to observe due to
> > autofluorescence though. Maybe use some PBS (or Ringer) with Glucose (wash
> > once) for GFP screening.
> > 
> > When you have some survivors colonies in a 10 cm dish after 2 weeks of G418
> > (leave out PenStrep during that time G418, is enough), seed 4 96 well plates
> > 1 cell per well (average) and keep two of them without G418. Then select for
> > colonies stable experssing GFP. (Maybe you want to enrich GFP expressing
> > cells before by preparative FACS before seeding.) 
> > 
> > Good luck, 
> > Wo
> > > 
> > > Hello,
> > > 
> > > re: problems with GFP expression
> > > 
> > > I cloned a fragement of DNA that contains 4 copies of NFkB responsive
> > > element-TK promoter-d2EGFP fusion from plasmid pNFkB-d2EGFP (sold by
> > > Clontech) into pd2GFP (again sold by Clontech) that has Neo marker in it. I
> > > linearized the plasmid by using an enzyme that cuts approximately 100 bases
> > > ahead of the NFkB responsive elements and transfected HepG2 (hepatocyte)
> > > cells. I then established a stable cell line that is resistant to 500 ug/mL
> > > G418 for several generations now. On inducing these cells with 100 ng/mL of
> > > IL1 for as long as 4-6 hours, I dont see any GFP fluorescence. Does anybody
> > > have any thoughts why it is not working? Thanks for any advice.
> > > 
> > > Murali Vemula
> > > 
> > > ___________________________________________________________________
> > > Murali Vemula, Ph.D
> > > Research Fellow
> > > Harvard Medical School/Shriners Hospital
> > > 51 Blossom Street
> > > Boston, MA 02114
> > > 617-371-4927 (phone)
> > > 617-371-4951 (fax)
> > > 
> > > ---
> > > 
> > > 
> > 
> > 
> > -----
> > Dr. Wolfgang Schechinger
> > Institute of Biomedical Sciences
> > Academia Sinica, Taipei, Taiwan
> > 
> > ---
> > 
> 
> ---
> 
> 


-----
Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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