over expression clone
bercollet at yahoo.fr
Wed Mar 10 10:41:31 EST 2004
> Hi everyone, I wanted to establish a over expression clone by
> transfected a gene to the cell line. Finally I got some stable clones
> after the drug selection. Surprisingly, I could not obtain any
> over-expressors. The gene expression level is even lower than the
> endogenous level comparing to the parental cells. Could anyone give an
> explanation for the phenomenon?
The insertion site of transgene may be located in the promoter ...
Did you use a circular plasmid to transfect or did you linearise the
construct before transfection ? If you linearise it you probably reduce
greatly the chance of having the insertion site within the promoter region.
How many clone did you screen ? As insertion in the genome occurs at random
(at least for conventional plasmids), you may have picked up a bad clone.
When you say the expression level is lower than the endogenous level, do you
mean that you have a way to distinguish the 2 (tag) ? Is it lower or not
existant at all ... in the latter case, your construct may be in cause (if
all the clones you sreened are in that way).
Dr Bertrand Collet
Scottish Fish Immunology Research Centre
b.collet at CAPabdn.ac.uk
uncap to email me
More information about the Methods