Hi
I want to create stable cell lines for membrane proteins of interest in inducible expression system. I wonder if anyone has used pTRE-tight-BI system to express two genes simultaneously in HEK293/HEK393-GnTI cells. The manufacture states that co transfection with linear plasmid is must. I wonder if any vector with Hygromycin Puromycin resistance is available for this purpose. Any suggestion about pTRE-tight-BI or any other effective system is welcome.
Thank you
Shanti Pal
Hi Shanti,
I have no experience with the pTRE-tight-BI system but looking at the specs I can comment this;
- There is no antibiotic selection marker on that plasmid. It seems that one of the MCS would have to be used for that. Alternatively, you could include a marker gene using an IRES or 2A element, behind one of the two genes-of-interest.
- The plasmid has to be used together with a specific Takara Tet-On or Tet-Off cell system, so 293T, 293S or 293S GnTI- cells won't give you inducible expression. This could perhaps be solved by transfecting into HEK293 another plasmid encoding the specific compatible Tet-On or Tet-Off protein. I don't know whether such a plasmid is available from Takara.
- Geneva biotech has the MultiMam Stable system that could be very suitable. https://geneva-biotech.com/product_category/mammalian-cell-expression/multimam-stable/
- We wrote a Nature protocols paper on using lentivirus to make stable cell lines that could be suitable. PMID: 30455477. Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9.
Cheers Jonathan
Hello Shanti,
I never used inducible system, but I have used a backbone with two promoters (CMV and EF1a) for cooverexpression, with Zeocin antibiotic selection (low concentration for selection). It is called pBudCE4.1 from Thermofischer.
https://www.thermofisher.com/order/catalog/product/V53220
Hope that helps.
https://www.addgene.org/vector-database/
Try also to check this site searching for your target vectors..
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