In article <3852E51B.2FB52C77 at po.cwru.edu>, Dennis Gor
<URL:mailto:dog at po.cwru.edu> wrote:
> Greetings
>> I need some help with transfection and expression of plasmids in B
> lymphocytes/ hybridomas. I have reporter plasmids that utilize the CMV
> enhancer / chicken b actin promoter tandem (pBac Mam-2 from Novagen,
> Inc.). I have no trouble detecting expression of the GFP or beta gal
> reporters in baby hamster kidney (BHK 21).
>> I am unable to detect expression in b cell lines, or primary spleen
> lymphocytes transfected under similar conditions using either lipofectin
> (BRL) or Effectene (Qiagen). I have also attempted stimulation of the b
> cells with LPs and/ or PMA. I have been looking for expression by fixing
> intact cells. I have not attempted to demonstrate expression in cell
> lysates?. Is my problem of detection one of lack of sensitivity?
>> Is the CMV enhancer appropriate for expression in lymphocytes?
> Is there a commercial source of plasmids that sponsor gene expression
> under control of the Immunoglobulin promoter/ enhancer tandem?
>> Alternatively does anyone have any thoughts on a suitable promoter
> system to use for expression of plasmids in B cells/ hybridoma's?
>> Please copy replies to my e-mail address. Thanks very much for any
> thoughts on this.
>> dennis gor <dog at po.cwru.edu>
>
CMV enhancer and a b-actin promotor should work I think.
There are a large number of vector systems which work in
hybridomas/plasmacytomas and which I guess should also work in primary
B-cells.
Just look at papers on the expression of recombinant antibodies which use
either "J558L", "NS0", "Y0", "YB2/0" as cell lines to be transfected. I
have always used as a vector system the pSV vectors originally modified by
Michael Neuberger to include the immunoglobulin intron enhancer.
Search for papers by Michael (1984 I think?) or later papers by M.
Bruggemann (1986/7) and G.Winter (1986/7 onwards) to get more details on
this vector.
Mike Clark, <URL:http://www.path.cam.ac.uk/~mrc7/>
--
o/ \\ // || ,_ o M.R. Clark, PhD. Division of Immunology
<\__,\\ // __o || / /\, Cambridge University, Dept. Pathology
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