B lymphocyte transfection and expression

Dennis Gor dog at po.cwru.edu
Sat Dec 11 18:28:09 EST 1999


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 expresssion 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 adress. Thanks very much for any
thoughts on this.

dennis gor <dog at po.cwru.edu>

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