Expression of chemokine receptor in RBL-2H3 cells
Louis de Leseleuc
deleseleuc at myrealbox.com
Tue Sep 10 20:38:37 EST 2002
So many things you can do, and some many things that can go wrong. Let's
1) Sometimes cell lines will not properly express proteins from gene
originating from other species, especially proteins that go through the
secretory pathway. They will get stuck in the golgi or endosomes or such
because the cell lack the proper targeting equipment. I have seen this
happen with monkey kidney cells trying to express human
hematopoietic-specific membrane proteins. Your human to rat strategy, as it
seems, could result in that. To see if it is the case, you can do some
immunofluorescence on stably or transiently transfected cells (don't try PE,
it bleaches). Or you can do FACS with permeabilized cells.
2) You can transietly transfect your plasmid in some easy high-yield cell
line like 293T and observe protein expression by FACS or Western/dot blot.
This helps you prove that your gene is "expressible", and that the protein
is not degraded.
3) Did you linearize you plasmid? This helps to generate optimal clones or
populations. Cut in the AmpR region.
That's a beginning. Good luck!
"Ming" <ming88us at yahoo.com> wrote in message
news:20020909173640.9512.qmail at web40514.mail.yahoo.com...
> Hi, there
> I'm working with some chemokine receptors and try to
> establish stable RBL-2H3 cell lines expressing them.
> The cDNAs were cloned into pcDNA3.1(ECoRI and XbaI)
> and the recombinant plasmid (digested with Bgl II) was
> transfected into RBL-2H3 cells by electroporation.
> After 3 weeks of selection with 800ug/ml G418, cells
> were pooled and stained with PE-conjugated monoclonal
> antibody to detect the receptor expression on the cell
> surface. Unfortunately we did not get any positive
> cells on FACS, although DNA sequencing and RT-PCR
> results were fine. BTW, the antibody was good, since
> it worked very well with human monocytes.
> What shall I do? Any suggestion is welcome.
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