The Tet-on 3G doxycycline-inducible expression system (#631354, Takara Bio USA) was used to induce the expression of shKDM4C inserted under the TRE3G promoter in U87 cells according to the manufacturer’s protocol. U87 cells were treated with lentivirus-Tet3G (pLVX-EF1a-Tet3G regulator vector) and selected with G418 (2 mg/ml) to generate U87-Tet3G cells. Next, U87-Tet3G cells were stably transfected with pLVX-TRE3G-shKDM4C (shKDM4C sequences; #1, NM_015061.1-1702s1c1, TRCN0000022058 and #2, NM_015061.1-768s1c1, TRCN0000022054) response vector and selected with puromycin (2 μg/ml) for Tet-On inducible shKDM4C cells. The expression of KDM4C was confirmed in media containing doxycycline (Dox; 100 μg/ml).
Tet on 3g doxycycline inducible expression system
Manufactured by Takara Bio
Sourced in United States
The Tet-on 3G doxycycline-inducible expression system is a genetic engineering tool designed to enable controlled gene expression in mammalian cells. It utilizes a modified tetracycline-responsive transcriptional activation system to induce target gene expression upon the addition of doxycycline to the cell culture medium.
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3 protocols using tet on 3g doxycycline inducible expression system
1
Doxycycline-Inducible Knockdown of KDM4C in U87 Cells
2
Inducible HP1γ Expression in SiHa Cells
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The Tet-on 3G doxycycline-inducible expression system (Takara Bio USA, #631354) was used to promote the expression of HP1γ inserted under the TRE3G promoter in SiHa cells according to the manufacturer’s protocol. After cloning HP1γ wild-type (WT) or AA mutant into the pLVX-TRE3G-IRES vector, we gathered lentiviral supernatants from 293T cells expressing both regulator vector (pLVX-EF1a-Tet3G) and response vector (pLVX-TRE3G-IRES-HP1γ WT or pLVX-TRE3G-IRES-HP1γ AA). Then, SiHa cells were coinfected with the two viruses following G418 selection (500 μg/ml). After incubation with or without doxycycline (1 μg/ml) for additional 48 h, the cells were harvested for analyses.
3
Inducible BAI1 Expression in Brain Tumor Cells
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The Tet-on 3G doxycycline-inducible expression system (Takara Bio, 631,339) was used to induce the expression of full-length ADGRB1 cDNA under the TRE3G promoter in MB (ONS76 and DAOY) and glioblastoma (LN229) cell lines according to the manufacturer's protocol. Briefly, 293 T cells were transfected with pLVX-CMV-Tet3G (regulator vector) or pLVX-TRE3G-BAI1 (response vector) along with psPAX2/pMD2.G packaging vectors by FuGene HD (Promega, E2311) to harvest lentiviral particles. MB and glioblastoma cells were treated with lentivirus-Tet3G (pLVX-CMV-Tet3G) and selected with G418 (2 mg/ml) to generate TET3G cells. TET-3G cells were then infected with viral particles from the response vector and selected with puromycin (10 µg/ml) for Tet-On inducible BAI1 cells. The expression of BAI1 was confirmed in media containing doxycycline (Dox; 1 μg/ml). For siRNA-mediated knockdown experiments, 70-90% confluent cells were cultured in a 6-well plate and siRNA transient transfection was performed with RNAiMAX (Invitrogen, 13,778,075) as prescribed. The following siRNAs were used: siBAI1#Exon-10 (Invitrogen, AM16708, ID:4161) targeting exon 10 and siBAI1#Exon-23 (Invitrogen, 4392420, ID: s1870) targeting exon 23 of ADGRB1 (NM_001702.2) and FAM-labeled negative control, #Scramble (Invitrogen, AM4620).
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