Hello all luciferase people,
I have just tried Bujard&Gossen's TetR
inducible expression system by assaying luciferase activity as a reporter.
I transiently transfected the two constructs into sub-confluent mouse
myoblast C2C12 in the presence or absence of Tc. Without Tetracycline(Tc)
I got 5 120 690 counts. With 30ng/ml Tc I got 1 158 711 counts and with
300ng/ml Tc I got 445 877 counts - at best a 10-fold repression. We rather
hoped for 4-5 logs of repression.
I have never done luciferase assays before and thus wonder whether either
a) 5 000 000 counts is outside the linear limits of this assay
b) 400 000 counts is a pretty low level of expression and the gene we hope
to express in this system will be expressed at very low levels. If both of these
possibilities are true then the true level of repression could be much higher
than 10-fold. I transfected 100 000 cell in 35mm plates. The cell were incubated
for two days after added DNA and were confluent when harvested. 5ug DNA in
total were used and Promega Transfectam was the agent used. All the cells
were washed in PBS and scraped into 100ul luciferase buffer. 100ul buffer
containing luciferin and 0.2mM ATP were injected in a Berthold luminometer.
GRGGTA at picr.cr.man.ac.uk