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Tet-On inducible system


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#1 sanjiun

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Posted 08 March 2013 - 12:55 AM

I'm currently using Tet-On inducible system to overexpress my (GOI) gene-of-interest.
After tranfecting my GOI into the cell line, I found that:
1. Gene expression is low (well, I didn't use the lentivirus system) -- makes no difference from other plasmid based expression.
2. Expression is higher when induce with higher dose of doxycycline. But at the same time, the toxicity effect of doxycycline causing the cell death.
(let's say I would like to see if my protein makes cells proliferate faster. How to measure the proliferation while a lot of cells are dying when I induce with dox?)

Not sure how many here using this system (or similar system). Do you guys encounter the problems above?
I was wondering if it is worth it to use this system as too much hurdles to overcome, balancing toxic effect and the expression level. Sigh Posted Image

#2 cellthetruth

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Posted 08 March 2013 - 05:17 AM

you are sure you use cell lines containing a transactivator? without this it doesnt work. anyway we use the system in HEK cells which we previously stably transfected with the transactivator vector and the system works very nice for us. i think it is worth to use the system in particular because you shouldnt have to use toxic levels of doxycycline, at least we dont.

good luck!!!

#3 sanjiun

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Posted 10 March 2013 - 07:23 PM

Yupe. I transfected my cell lines with transactivator vector. The expression increased with higher doxycycline induction. But at the same time I see a lot of dead cells floating.
This is why I'm wondering. If the transfection didn't work, I shouldn't be seeing an increase in expression.
But my GOI is suppose to increase proliferation. Instead of cell growing, I see cells floating.
Weird...

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