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Lenti x bicistronic expression system

Manufactured by Takara Bio
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The Lenti-X Bicistronic Expression System is a lentiviral vector system designed for the efficient co-expression of two genes of interest. The system utilizes a bicistronic mRNA expression strategy to produce two distinct proteins from a single transcript. This streamlined approach allows for the simultaneous expression and evaluation of multiple genes in a variety of cell lines and applications.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Ptdtomato vector, by Takara Bio (2 mentions) Lapatinib, by Selleck Chemicals (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Mccoy s 5a, by Thermo Fisher Scientific (1 mentions) Laminin 5, by Merck Group (1 mentions) Plvx puro, by Takara Bio (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Transwell plate, by BD (1 mentions) Abi prism 7700 sequence detection system, by Thermo Fisher Scientific (1 mentions) Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions) Opti mem medium, by Thermo Fisher Scientific (1 mentions) Matrigel, by BD (1 mentions) G418 sulfate, by Harvard Bioscience (1 mentions) P tdtomato, by Takara Bio (1 mentions) Geneticin, by Harvard Bioscience (1 mentions)

Close spelling variants of this product

Lenti-X expression system Lenti-X

6 protocols using lenti x bicistronic expression system

1

Lentivirus Production and Cell Selection

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The Lenti-X Bicistronic Expression System from Clontech was used for lentivirus production according to the manufacturer’s instructions. Infection was performed in the presence of polybrene (8 mg/ml), and the cells were centrifuged at 1200 g for 60 minutes at 32°C. Infected cells were selected with puromycin (2.5 mg/ml).
Boulbes D.R., Arold S.T., Chauhan G.B., Blachno K.V., Deng N., Chang W.C., Jin Q., Huang T.H., Hsu J.M., Brady S.W., Bartholomeusz C., Ladbury J.E., Stone S., Yu D., Hung M.C, & Esteva F.J. (2014). HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer. Molecular Oncology, 9(3), 586-600.
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2

Investigating Akt/Erk Signaling in Cancer Cells

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Reagents were obtained from the following sources: lapatinib from Selleck Chemicals; Dulbecco's modified Eagle's (DMEM)/F12, McCoy’s 5A, RPMI, and fetal bovine serum (FBS) from Life Technologies; antibodies to phospho-T308 and phospho-Ser473 Akt, Akt, phospho-Erk1/2, Erk1/2, and cleaved-caspase 3 from Cell Signaling Technologies; laminin V from Sigma; HER2 from EMD Biosciences; c-myc (9E11) from Santa Cruz Biotechnology; and Alexa-conjugated antibodies from Invitrogen. The lentiviral expression plasmid pLVX puro and Lenti-X Bicistronic Expression System were purchased from Clontech.
Boulbes D.R., Arold S.T., Chauhan G.B., Blachno K.V., Deng N., Chang W.C., Jin Q., Huang T.H., Hsu J.M., Brady S.W., Bartholomeusz C., Ladbury J.E., Stone S., Yu D., Hung M.C, & Esteva F.J. (2014). HER family kinase domain mutations promote tumor progression and can predict response to treatment in human breast cancer. Molecular Oncology, 9(3), 586-600.
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3

Evaluating CXCR4 in Colon Cancer

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Mouse monoclonal anti-human CXCR4 antibody, GAPDH, RPMI-1640, fetal bovine serum (FBS), penicillin, streptomycin, Polybrene, phosphate-buffered saline (PBS; Hyclone, Logan, UT, USA), Matrigel (BD Biosciences, San Jose, CA, USA), RNA extraction kit, reagents for reverse transcription, western blotting kit (Boster Biological Technology Co., Wuhan, China), human colon cancer cell line (SW480 cells), nude (nu/nu) BALB/c mice (Shanghai Cancer Institute, Shanghai, China), Lenti-X Bicistronic Expression System (Clontech, Palo Alto, CA, USA), Lipofectamine 2000, Lipofectamine™ RNAiMAX transfection reagents (Invitrogen Corp., Carlsbad, CA, USA), Opti-MEM medium (Gibco-BRL, Gaithersburg, MD, USA), CellTiter96AQ cell proliferation detection kit (Promega, Madison, WI, USA), Transwell plates (BD), SYBR Green PCR Master Mix (Toyobo Biologics, Osaka, Japan), and thermal cycler (ABI PRISM® 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA) were used in the present study. This study was approved by the ethics board at the First Affiliated Hospital of Sun Yat-sen University.
WANG T.B., HU B.G., LIU D.W., SHI H.P, & DONG W.G. (2014). The influence of lentivirus-mediated CXCR4 RNA interference on hepatic metastasis of colorectal cancer. International Journal of Oncology, 44(6), 1861-1869.
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4

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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5

Generation of tdTomato and EpCAM-Expressing LL/2 Cells

Cited 2 times
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A PCR product containing the sequence of tdTomato (vector ptdTomato; #63-2531, TaKaRa Clontech) was cloned into the lentiviral expression vector pLVX-IRES-neo (LentiX-Bicistronic Expression System; #63-2181, TaKaRa Clontech) to generate a pLVX-tdTomato-IRES-Neo construct. Notably, a resistance-sequence for G418-sulfate is contained in the lentiviral expression vector. The resulting nucleotide pLVX-tdTomato-IRES-Neo was verified by Sanger sequencing and restriction enzyme digestion.12 (link) LL/2 was transfected with pLVX-tdTomato-IRES-Neo using lipofection (Lipofectamine 3000; Thermo Fisher Scientific). tdtLL/2 was enriched by cultivation in selection medium containing G418-sulfate (#A2912; Biochrom) and by repetitive FACS sorting. As previously described in detail,13 (link) tdtLL/2 cells were stably transduced with a pMXs vector containing the full-length murine EpCAM (UNIPROT entry: #Q99JW5) cDNA to generate the EpCAM-overexpressing cell line EpCAM/tdtLL/2.
Xu T., Karschnia P., Cadilha B.L., Dede S., Lorenz M., Seewaldt N., Nikolaishvili E., Müller K., Blobner J., Teske N., Herold J.J., Rejeski K., Langer S., Obeck H., Lorenzini T., Mulazzani M., Zhang W., Ishikawa-Ankerhold H., Buchholz V.R., Subklewe M., Thon N., Straube A., Tonn J.C., Kobold S, & von Baumgarten L. (2023). In vivo dynamics and anti-tumor effects of EpCAM-directed CAR T-cells against brain metastases from lung cancer. Oncoimmunology, 12(1), 2163781.
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6

Generating tdTomato-expressing LL/2 cells

Cited 1 time
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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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