over expression clone

Bertrand Collet 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 ...
disrupting it.
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).

Bertrand


-- 
Dr Bertrand Collet
Scottish Fish Immunology Research Centre
http://www.abdn.ac.uk/sfirc/
b.collet at CAPabdn.ac.uk
uncap to email me





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