Lenti x tet on advanced system

Manufactured by Takara Bio

Sourced in United States

The Lenti-X Tet-On Advanced System is a lab equipment product developed by Takara Bio. It is a tetracycline-inducible lentiviral expression system that enables tight, efficient, and reversible gene expression regulation in a wide range of cell types, including primary cells and stem cells.

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Lab products found in correlation

Hek293t, by American Type Culture Collection (1 mentions) Hydrocortisone, by Merck Group (1 mentions) Jetprime, by Polyplus Transfection (1 mentions) Puromycin, by Merck Group (1 mentions)

Spelling variants of this product

Lenti-X (77 citations) Lenti-X Tet-On advanced inducible expression system (17 citations) Tet-on system (11 citations) Tet-on Advanced System (6 citations) Lenti-XTM Tet-On® Advanced Inducible Expression System (4 citations)

3 protocols using lenti x tet on advanced system

Cell Culture and Transfection Protocols for Various Cancer Cell Lines

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MDA-MB-231 (ATCC HTB-26), PC-3 (ATCC CRL-1435) and HEK293T (ATCC CRL-11268) cells were maintained at 37°C in a humidified 10% CO₂ incubator, HNSCC61 cells were grown at 5% CO₂. All cells (except PC-3) were grown in DMEM. We obtained the HNSCC61 cells from Prof. Dr. Weaver [30 (link)] (Vanderbilt University) in August 2011, who obtained the cells from Prof. Dr. Yarbrough [31 (link)–33 (link)]. PC-3 cells were grown in RPMI. All media (from Gibco Life Technologies (Grand Island, NY, USA) were supplemented with 10% foetal bovine serum, 10 μg/mL streptomycin and 10 IU/mL penicillin. As an exception, HNSCC61 required 20% fetal bovine serum and an extra addition of 0.4 μg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA).
JetPrime (Polyplus Transfection Inc., New York, NY, USA) was used to perform transient expression according to the manufacturer’s protocol. For virus production HEK293T cells were transfected using the calcium phosphate method as described before [28 (link)]. Inducible stable cell lines were obtained by transduction of the VCA Nbs in the pLVXTP vector, making use of the Lenti-X Tet-On advanced system (Clontech, Mountain View, CA, USA) according to the manufacturer’s protocol. Expression was induced by addition of 500 ng/mL doxycycline.

Hebbrecht T., Van Audenhove I., Zwaenepoel O., Verhelle A, & Gettemans J. (2017). VCA nanobodies target N-WASp to reduce invadopodium formation and functioning. PLoS ONE, 12(9), e0185076.

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Engineered Lentiviral System for Neuronal Conversion

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The Lenti-X Tet-On Advanced System (Clontech, Mountain View, CA, USA) was modified by replacing the CMV promoter of the pLVX-tetOn Advanced vector with the EF1α promoter, resulting in the pLVX-EtO vector.³² (link) Coding sequences for the neuronal conversion TFs were synthesized and cloned into the pLVX-Tight-Puro construct (Clontech). The pro-neuronal factors ASCL1 AN were linked by a 2A peptide sequence or target TF sequences based on human ORF before insertion into the pLVX-Tight-Puro vector. The lentiviral vector plasmid, the packaging plasmid, and the envelop plasmid were co-transfected into HEK293FT cells using polyethyleneamine. Resulting viral particles were enriched by ultracentrifugation. They were then frozen for future use.³² (link)

Hirayama M., Mure L.S., Le H.D, & Panda S. (2024). Neuronal reprogramming of mouse and human fibroblasts using transcription factors involved in suprachiasmatic nucleus development. iScience, 27(3), 109051.

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Lentiviral Ephrin-B2 Overexpression

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For constitutive overexpression, the Lenti-X Tet-On Advanced System (Clontech, Mountain View, CA) was modified by replacing the cytomegalovirus promoter of the pLVX-Tet-On plasmid with the elongation factor 1 (EF1) α promoter, resulting in the pLVX-EF1α vector.
The pLVX-EF1α vector was a generous gift from Dr. Jerome Mertens (University of Bonn).
Human ephrin-B2 full-length (NM_004093.3) or fragments closely resembling the Cterminal fragment (CTF) or intracellular domain (ICD) were amplified by PCR. PCR products were restricted with MluI and BamHI and ligated into the pLVX-EF1α vector.
Production of lentiviral particles was performed as previously described (Kutner et al. 2009) .
Briefly, HEK-293FT cells were co-transfected with the packaging plasmid psPAX2, the envelope plasmid pMD2.G, and the respective lentiviral vector plasmid. Viral particles were enriched by incubation with polyethyleneglycol 6000 and consecutive centrifugation.
Presenilin expressing and WT ESdM were transduced with the different ephrin-B2 constructs overnight. After antibiotic selection, cells were cultured in the presence of 2 μg/mL puromycin (Sigma-Aldrich).

Kemmerling N., Wunderlich P., Theil S., Linnartz-Gerlach B., Hersch N., Hoffmann B., Heneka M.T., de Strooper B., Neumann H, & Walter J. (2017). Intramembranous processing by γ-secretase regulates reverse signaling of ephrin-B2 in migration of microglia. Glia, 65(7).

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