Ptdtomato vector

Manufactured by Takara Bio

Sourced in United States, France

The PtdTomato vector is a plasmid DNA construct containing the gene for the TdTomato fluorescent protein, which is derived from the tandem dimer Tomato protein. The vector is designed for expression of the TdTomato protein in cells or organisms.

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Lab products found in correlation

Lenti x bicistronic expression system, by Takara Bio (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Gene pulser xcel electroporator, by Bio-Rad (1 mentions) Facscanto 2 flow cytometer, by BD (1 mentions) Geneticin, by Harvard Bioscience (1 mentions)

4 protocols using ptdtomato vector

Engineering tdTomato-expressing Salmonella

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To generate tdTomato-expressing S. typhimurium (VNP-tdT), the ptdTomato vector (Clontech) was electroporated into the S. typhimurium strain VNP20009 (obtained from American Type Culture Collection, Manassas, VA, USA) using a Genepulser Xcel electroporator (Bio-Rad, Hercules, CA, USA) at 2.5 kV, 1,000 Ω. VNP-ctrl, a control transfectant S. typhimurium, was generated by transfection of the vector plasmid without tdTomato. Transformed S. typhimurium were grown in modified Luria–Bertani medium containing ampicillin (100 µg/mL) until the late log phase and then diluted in DMEM before administration to the mammalian cell cultures. To calculate colony-forming units (CFU)/mL, the following conversion was used: OD₆₀₀ of 1 = 10⁹ CFU/mL. After coculturing the tumor cells and bacteria, the extracellular bacteria were removed using kanamycin (100 µg/mL). To confirm infection efficiency, the percentage of tdTomato⁺ B16F10 cells was evaluated using a FACSCanto II flow cytometer (BD Biosciences, San Diego, CA, USA).

Horiuchi Y., Nakamura A., Imai T, & Murakami T. (2024). Infection of tumor cells with Salmonella typhimurium mimics immunogenic cell death and elicits tumor-specific immune responses. PNAS Nexus, 3(1), pgad484.

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NF-κB Responsive Fluorescence Reporter

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An NF-κB responsive red-fluorescence reporter was generated by replacing the CMV-promoter in the ptdTomato vector (Takara/Clontech; Saint-Germain-en-Laye, France) with an artificial promoter composed of five NF-κB consensus binding sites, introduced via synthetic oligonucleotides at the 5`end of a cytomegalovirus (CMV) minimal promoter fragment. Expression vector constructs for N-terminally EGFP-tagged wildtype or mutant p105 or p50 (300ng per well each) and non-tagged RelA (5ng per well if not indicated otherwise) were transfected together with the reporter gene vector (100ng per well) into HEK293T cells grown on collagenized 48-well plates. Non-transfected, reporter-only and reporter-plus-RelA-only samples were included as controls. Fluorescence intensities were determined in live cells using a FluoroSpot Analyzer (CTL Immunospot, Bonn, Germany) with separate recordings of the tdTomato (red) and the EGFP (green) signals in each well. Plates were scanned repeatedly within 42-48 and 66-72 hours after transfection using variable magnifications and exposure settings. Fluorescence values were quantified with ImageJ (30 (link)) and normalized to the “reporter only” baseline control which was defined as 1-fold.

Fliegauf M., Kinnunen M., Posadas-Cantera S., Camacho-Ordonez N., Abolhassani H., Alsina L., Atschekzei F., Bogaert D.J., Burns S.O., Church J.A., Dückers G., Freeman A.F., Hammarström L., Hanitsch L.G., Kerre T., Kobbe R., Sharapova S.O., Siepermann K., Speckmann C., Steiner S., Verma N., Walter J.E., Westermann-Clark E., Goldacker S., Warnatz K., Varjosalo M, & Grimbacher B. (2022). Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50. Frontiers in Immunology, 13, 965326.

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Cloning tdTomato into Lentiviral Vector

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A PCR product containing the coding sequence of tdTomato (vector ptdTomato; catalog no. 632531; TaKaRa Clontech) was cloned into the lentiviral expression vector pLVX-IRES-neoR (Lenti X Bicistronic Expression System; catalog no. 632181; TaKaRa Clontech). The resulting construct pLVX-tdTomato-IRES-Neo was verified by restriction enzyme digest and Sanger sequencing.

Mulazzani M., Fräßle S.P., von Mücke-Heim I., Langer S., Zhou X., Ishikawa-Ankerhold H., Leube J., Zhang W., Dötsch S., Svec M., Rudelius M., Dreyling M., von Bergwelt-Baildon M., Straube A., Buchholz V.R., Busch D.H, & von Baumgarten L. (2019). Long-term in vivo microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice. Proceedings of the National Academy of Sciences of the United States of America, 116(48), 24275-24284.

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Generating tdTomato-expressing LL/2 cells

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A vector was generated by cloning a PCR-product containing the tdTomato-sequence (vector ptdTomato; #632531, TaKaRa Clontech) into the lentiviral expression vector pLVX-IRES-neo (LentiX-Bicistronic Expression System; #632181, TaKaRa Clontech) [19] (link). This lentiviral expression vector contains a resistance-sequence for G418-sulfate. The resulting nucleotide construct pLVX-tdTomato-IRES-Neo was verified by restriction enzyme digestion and direct sequencing.
LL/2-cells were transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). Immediately after transfection, ^tdtLL/2-cells were cultured in selection medium containing G418-sulfate and Geneticin (#A2912; Biochrom). TdTomato-positive clones were further enriched using FACS sorting [19] (link).

Zhang W., Karschnia P., von Mücke-Heim I.A., Mulazzani M., Zhou X., Blobner J., Mueller N., Teske N., Dede S., Xu T., Thon N., Ishikawa-Ankerhold H., Straube A., Tonn J.C, & von Baumgarten L. (2021). In vivo two-photon characterization of tumor-associated macrophages and microglia (TAM/M) and CX3CR1 during different steps of brain metastasis formation from lung cancer. Neoplasia (New York, N.Y.), 23(11), 1089-1100.

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