Northern blot analysis was carried out as previously described.22 (link),23 (link) γ32P-ATP-labeled oligonucleotides complementary to miRNAs were used as probes. U6 snRNA was used as a control for quantification. The stability of uridylated pre-let-7 was assessed using the Tet-Off Advanced Inducible Gene Expression System (Clontech). pTRE-Tight-pri-let-7g was constructed by inserting pri-let-7g into pTRE-Tight vector (Clontech). HepG2 cells with pTet-off Advanced vector (Clontech) were transfected with pTRE-Tight–pri-let-7g and control siRNA or siRNA for MCPIP1. At 48 h post-transfection, pri-let-7g transcription was halted by addition of doxycycline (DOX) (1 μg/mL). Total RNA was extracted at the indicated time periods and subjected to northern blot analysis.
Ptet off advanced vector
Manufactured by Takara Bio
The PTet-off Advanced vector is a plasmid designed for regulated gene expression in mammalian cell lines. It contains the tetracycline-controlled transactivator (tTA) under the control of a constitutive promoter, allowing for tight regulation of target gene expression.
Automatically generated - may contain errors
2 protocols using ptet off advanced vector
1
Assessing Uridylated pre-let-7 Stability
2
Inducible Gene Expression in LNCaP Cells
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LNCaP cells were transfected with pTet-Off Advanced vector (Clontech). A stable transformant was isolated by 800 μg/ml G418 (Wako), and tested for induction according to the manufacturer’s protocol. LNCaP tet-off cells were maintained in RPMI1640 medium with 10% FCS containing 400 μg/ml G418 and 1 μg/ml doxycycline (Clontech). LNCaP tet-off cells were transfected with pTRE2hyg-FLAG-AIbZIP, selected using 1 mg/ml hygromycin B (Wako), and maintained in RPMI 1640 medium with 10% FCS containing 400 μg/ml G418, 500 μg/ml hygromycin B and 1 μg/ml doxycycline.
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