I am looking to use a tet expression system to express my GOI in HK2 and mIMCD3 cells. I was going to go with the two vector tet-on system by Clonetech, but now I see they actually have an expression system that expresses both the GOI and Tet3G protein on the same vector (Tet-One). I was wondering if anyone has experience with this or know if one system is better than another system. I saw there is also a Tet-express system where the vector doesn't actually express the Tet transcription factor, instead, it is added to the media. If you have any insight into which vector system you prefer and why it would be greatly appreciated. I imagine using a 1 vector system saves time because you do not need to create doubly-stable lines, but I don't know if that ends up affecting your GOI expression levels at all. TIA!
I use the Clonetech system for many years. With the tet-off you will get the highest induction (for our last cell line it is 2000-10000 fold.) Several critical points: 1) you will have to do the 2 stable rounds of transfection (what in fact might be a benefit also as you can freeze the first stable cell line and do second round for any gene of interest forever). 2) You will need a good batch of doxycycline to repress your gene under baseline conditions. 3) With the induction levels you can reach, it is difficult to answer whether this is really a meaningful biological response.
I have found out that I am unable to use the 2 vector system because one of out cell lines is G418 resistant. I think I'm stuck with the one vector system now because that only requires pur resistance. Anyone have experience with the 1 vector system? It seems that this system is relatively new. I'm just curious if you ended up linearizing your vector before transfection and the approx. fold induction with dox?
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