To assess the effect of Kl on the proliferation and differentiation of HPCOA07/03 cells, these cells were genetically engineered to conditionally overexpress the secreted form of KL using the Lenti-XTM Tet-On® Advanced Inducible Expression System (Clonetech). Gene expression is activated in this system using the tetracycline Doxycycline. The Lenti-XTM Tet-On® Advanced Inducible Expression System consists of a regulator vector, pLVX-Tet-On Advanced, and a response vector, pLVX-Tight-Puro. The regulator vector constitutively expresses a tetracycline-controlled transactivator (rtTA-Advanced) that, in the presence of Doxycycline, binds to the inducible promoter (Ptight) of the gene of interest in the response vector and activates transcription. Ptight consists of a tet-responsive element joined to a minimal CMV promoter. Induction of the system produces high-level transcription of the gene of interest.
Lenti xtm tet on advanced inducible expression system
Manufactured by Takara Bio
The Lenti-X™ Tet-On® Advanced Inducible Expression System is a lentiviral-based tool for regulated gene expression in mammalian cells. It allows for the tight control of target gene expression in a dose-dependent manner using the tetracycline-responsive promoter system.
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4 protocols using lenti xtm tet on advanced inducible expression system
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Conditional Overexpression of Secreted KL
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Conditional KL Overexpression in HPCOA07/03 Cells
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Generating Cell Lines for Myc and Ras Studies
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To prepare BJ fibroblasts stably expressing C‐Myc fused to the modified estrogen receptor ligand‐binding domain (MycER, (Littlewood et al., 1995 )), pBabe puro‐MycER (provided by T. Littlewood) or pBabe puro plasmids were transfected into Phoenix packaging cells (from G. Nolan, Stanford). Obtained retroviral supernatants were filtered and used with 10 μg/ml of polybrene for 3 rounds of 24‐h infections. Transduced cells were selected using 1 μg/ml of puromycin for 3 days and then grown in Dulbecco's modified Eagle's medium without phenol red (DMEM/F‐12) (Gibco) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin and 0.5 μg/ml of puromycin. MycER activation was induced by 100 nM of 4‐hydroxytamoxifen (4‐OHT) dissolved in ethanol. U2‐OS MycER cells were kindly provided by Martin Eilers lab (University of Wuerzburg).
BJ human fibroblasts with doxycycline (Dox) inducible expression of H‐RasV12 (Ras) (Lenti‐XTM Tet‐On Advanced Inducible Expression System, Clontech) were prepared by double lentivirus infection and subsequent selection as described previously (Evangelou et al., 2013 ). Cells (estimated population doubling time between 35 and 45 h) were cultured in DMEM with 10% FBS, penicillin, streptomycin, 0.5 μg/ml of puromycin and 100 μg/ml of G418. Ras overexpression was induced using 2 μg/ml of Dox.
BJ human fibroblasts with doxycycline (Dox) inducible expression of H‐RasV12 (Ras) (Lenti‐XTM Tet‐On Advanced Inducible Expression System, Clontech) were prepared by double lentivirus infection and subsequent selection as described previously (
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Inducible Cell Line Generation Protocol
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BJ human fibroblasts (ATCC), U2-OS MycER cells (kindly provided by Martin Eilers, University of Würzburg) and the Phoenix–Ampho retroviral packaging cell line (kindly provided by Garry Nolan, Stanford University, CA, USA) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml of penicillin and 50 mg/ml of streptomycin, and for Phoenix–Ampho cells in addition 1 mM sodium pyruvate. BJ cells with doxycycline (DOX)-inducible expression of RasV12 (Lenti-XTM Tet-On Advanced Inducible Expression System, Clontech) (BJ-Ras) were created by double lentivirus infection as previously described [35 ], and were maintained in culture media as above containing 0.5 μg/ml of puromycin (Sigma, P8833) and 100 μg/ml of G418 (Clontech, 631307). RASV12 expression was induced using 2 μg/ml of doxycycline (Sigma, D9891). BJ-MycER and BJ-Ras/MycER cells were cultured in DMEM medium without phenol red (Gibco) supplemented with 10% FBS and antibiotics. MycER [37 ] activation was induced by 50–200 nM of 4-hydroxytamoxifen (4-OHT) (Sigma) dissolved in ethanol (Kemetyl).
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