Tet inducible expression

Graham Atherton grggta at picr.cr.man.ac.uk
Thu Oct 26 03:56:30 EST 1995


We have used this syste and found it is possible to routinely get 20-50x
repression in the cell lines we use - Mouse muscle myoblast, 3T3, B cell
lines eg Daudi. Maximum expression seems quite high going by luciferase
reporter assay but repressed expression has never been as low as the
authors suggested in their original paper. This is apparently down to 
cell type specificity of the system - I think they used HeLa or some
such and got up to 5 log's repression! A recent paper discusses this point
- I can't remember where it was published but a search of this years
publications should pick it up - they tried the system in several cell
lines and found variability in the degree of repression it was possible to 
get.
A more serious problem for us has been it is difficult to generate stable
cell lines with the system (this could be down to operator error on our
part - my boss did the experiments and he is a molecular biologist rather
than a tissue culture expert). We were unable to generate a line which
expressed our gene of interest without Tet. This experiment was not tried
with tet as far as I know leaving the possibility open that our gene is 
toxic at high levels of repression - though we have generated lines
stably expressing the same gene. Different cell line, lower levels of
expression using a different expression construct???
Please anyone let me know if the have similar problems, any solutions
with this system.
Cheers
Graham



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