I am planning to make inducible sh cell line for E14 cells. I am going to order either TRIPZ plasmid or Addgene's Tet-pLKO-puro plasmid. I am wondering if anyone could give me some feedback about the success ratio of these plasmids.
Thank you very much for your answers in advance.
Hi Aysegul,
I have used both pTRIPZ as well as pLKO-Tet ON- Puro Plasmid in mouse ES Cells and providing you first hand feedback. Even though pTRIPZ has good features like inducible system and RFP, I have always had trouble in able to successfully package pTRIPZ in a virus. Its a huge 13kb plasmid. Besides, I have heard from other users that the ability of this plasmid to be recognised by the miRNA machinery to form the hairpin is poor.
pLKO on the other hand has always been a favourite. Even though it lacks GFP/RFP, as long as it has one marker for selection (Puro), it's alright. This works beautifully. You'll just need to titrate the amount of Doxycycline.
The biggest disadvantage of the Tet inducible system is that you have to culture your cells in a Dox/Tet free medium. If Serum used in the culturing of cells is not Tet free, you will observe leaky expression of the shRNA and see knockdown before you really want it. In order to overcome this issue, Sigma made another plasmid called pLKO-3xLaco (http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Vector/1/plko_iptg_3xlaco.pdf). This is inducible with IPTG which is never a serum component and can be added to any culture medium.
Hope this information is helpful. Good Luck!
Utkarsh
Dear Utkarsh,
Thank you very much for your very detailed answer!! It has been really helpful.
I already borrowed TRIPZ plasmid from another lab and I guess I will start working with this plasmid. But I want to have a back up plan. That's why I asked Addgene plasmid which is quite cheaper compared to other available plasmids. My only concern about pLKO-tet-on was that I read a lot of negative feedback about the plasmid. After your answer, I am thinking of trying the both plasmid at the same time.
For mouse ES cells plko-tet-on works much better than any other plasmids
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