Hi
You mention the strain is AX2-GFP. How do wild type AX2 cells behave
during the chemotaxis assay? Is this a stable GFP strain such that the GFP
has integrated randomly into the genome? If yes, as already mentioned, the
integration may have created an aggregation-minus mutant.
Second, in my experience, and maybe others have also seen this without
reporting it, but GFP expression in Dicty is not always a benign event and
clones often need to be screened for GFP expression, proper growth and
developmental timing. I have transformed cells with pTX-GFP (different AX3
strains from the dictyBase) and AX4 cells, and have noticed a proportion
of GFP-positive clones do not behave like the non-transformed parent
including changes in developmental timing. I have not explored why, but
just assumed it was likely a copy number effect that imparts a dominant
negative phenotype. However, I have not seen this sort of thing with lacZ
markers.
Third, I find a number of GFP-transformed cells show a mild enrichment of
staining in the nucleus...maybe others have seen this too. Again, this
could be problematic with respect to nuclear function and gene expression.
It's speculative, but I believe too much GFP is not without consequence.
Lastly, is the cAMP being prepared properly in Soerensen's buffer (e.g, pH
~ 6.5) before being applied to the agar?
Best,
Mike
Sarah Power wrote:
>
> Hi,
>
> I want to do chemotaxis experiences with dictyostelium (strain Ax2-GFP).
> I tried with the protocols on the website of DictyBase. For exemple, I
> tried 1% and 2% agar in Sörensen's buffer an I used cAMP at differents
> concentrations: 10mM, 1mM, 200uM, 100uM, 10uM, 1uM. I did wells in a 6
> hole-plate(fill with agar) and added 15 ul of cAMP. I put a drop of 1ul
> of dicty at 2, 4, and 6mm from the edge of the well and I waited 3,4 or 5
> hours before imaging the cells.
> For the preparation of the cells, I tried a 5 or 6 hours starvation
> period at 10 000 000 cells/ml than I resuspend cells at 250 000 000
> cells/ml, I also tried an overnight starvation at 18oC and a period of
> three hours at 22oC before resuspending cells at 250 000 000 cells/ml.
> Despite all my effort, the cells do not seem to move towards the gradient
> of cAMP as it is suppose to be.
> It's important for my further experiences to use cAMP for chemoattractant,
> therefore I do not want to use folate.
>
> Do you know anything that could help me?
>
> Thank you
>
> Sarah Power
>
>
----------------------------------------
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
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