Ptripz lentiviral vector

Manufactured by Horizon Discovery

Sourced in United States

The PTRIPZ lentiviral vector is a tool used for gene expression and regulation in various cell lines. It functions as a viral delivery system to introduce genetic material into target cells. The PTRIPZ vector contains elements necessary for packaging, transduction, and regulated expression of transgenes.

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Lab products found in correlation

Pcmv vsv g, by Addgene (2 mentions) Pmscv ires gfp, by Addgene (1 mentions) Doxycycline, by Merck Group (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Pcmv dr8.2 dvpr, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions)

4 protocols using ptripz lentiviral vector

CRISPR-Mediated Gene Manipulation in Cells

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A single FLAG-tag was added to the N-terminus of the cDNAs of yeast ASP1 or zebrafish zASPG. The cDNAs were inserted into a modified pTRIPZ lentiviral vector (Dharmacon) or pMSCV-IRES-GFP (Addgene). Guide RNAs against human glutamine synthetase (sgGLUL-1, sgGLUL-2 and sgGLUL-3) were cloned into pLentiCRISPR V2 vector. For CRISPR rescue experiment, mouse Glul cDNA was cloned into pTURN-hygro retroviral expression vector, and HA tag was added to the C-terminus. Human ASRGL1 cDNA was cloned into pMSCV-puro retroviral expression vector (Clontech). Lentiviral particles were produced in 293T cells by using psPAX2 and pMD.2 packaging plasmids (Addgene). Retroviral particles were produced in 293T cells by using pcleo and pCMV-VSV-G packaging plasmids (gift from Scott Lowe). Cells were infected with viral supernatant in presence of 6 μg/mL of polybrene overnight and subjected to puromycin (2 μg/mL) or hygromycin (200 μg/mL) selection, or GFP sorting.

Pavlova N.N., Hui S., Ghergurovich J.M., Fan J., Intlekofer A.M., White R.M., Rabinowitz J.D., Thompson C.B, & Zhang J. (2018). As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid. Cell metabolism, 27(2), 428-438.e5.

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Inducible SLX4 Knockdown in HEK293A

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SLX4 shRNA sequences were inserted into a pTRIPZ lentiviral vector (Dharmacon, Lafayette, CO, USA). HEK293A cells were infected with a supernatant containing the virus and selected with 2 μg/ml puromycin. To induce knockdown (KD) of SLX4 protein, we added 1 μg/ml doxycycline (DOX) to cells for 48 h.
ishSLX4-84: AGGAGAAAGGAAGACACAA
ishSLX4-85: TGGAGCTAGAACAAACCAA

Zhang H., Chen Z., Ye Y., Ye Z., Cao D., Xiong Y., Srivastava M., Feng X., Tang M., Wang C., Tainer J.A, & Chen J. (2019). SLX4IP acts with SLX4 and XPF–ERCC1 to promote interstrand crosslink repair. Nucleic Acids Research, 47(19), 10181-10201.

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Lentiviral Knockdown of Oncogenes in Lung Cancer

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Lentiviral particles were produced in HEK 293 T/17 cells by co-transfection of lentiviral packaging plasmids pCMV-VSVG and pCMV-dR8.2 dvpr (Addgene) with pTRIPZ lentiviral vectors (GE Dharmacon) expressing the desired shRNAs according to the manufacturer’s protocol. The following pTRIPZ lentiviral vectors were used: a lentiviral vector expressing a non-targeting control shRNA (shCtrl, RHS 4743), two lentiviral vectors expressing different shRNAs targeting KRAS (shKR#1, V3THS_314004 and shKR#2, V3THS_314009), two lentiviral vectors expressing different shRNAs targeting Aurora A (shAKA#1, V2THS_12364 and shAKA#2, V2THS_153609) and two lentiviral vectors expressing different shRNAs targeting Aurora B (shAKB#1, V2THS_28606 and shAKB#2, V2THS_28601). In each case, A549 and H358 cells were infected with 1 MOI of lentiviral particles and selected with 2 μg/mL puromycin (#A1113803, Life Technologies) for two weeks. Individual cell clones were induced with 2 μg/mL doxycycline (#D9891, Sigma-Aldrich) for 5 days and screened for expression of the reporter red fluorescent protein (turboRFP), as well as for inhibition of expression of shRNA targets. Cells with at least 80 % knockdown levels were used to perform all biological assays.

dos Santos E.O., Carneiro-Lobo T.C., Aoki M.N., Levantini E, & Bassères D.S. (2016). Aurora kinase targeting in lung cancer reduces KRAS-induced transformation. Molecular Cancer, 15, 12.

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Inducible Lentiviral System for EMT-TF and shRNA

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Dox-inducible pLUT-zeo or -puro lentiviral vector (Suppl.Fig.9) was used for overexpression of EMT-TFs ORFs and microRNA genes. Constitutive pGIPZ or Dox-inducible pTRIPZ lentiviral vectors (Dharmacon/Horizon) were used for expression of shRNAs, see Suppl.Table2 for details. Briefly, lentiviral particles were packaged in HEK293T cells (RRID:CVCL_0063) following calcium phosphate cotransfection of transfer vector, psPAX2 (Addgene, 12260), and pCMV-VSV-G (Addgene, 8454). After 2-4 rounds of infection with lentiviral particles, cells were selected in zeocin (50 μg/ml) or puromycin (1 μg/ml) containing media for ten days. Mass culture pools representing hundreds of drug resistant clones were used in all experiments. Double expressing cell lines were established by superinfection with the second shRNA vector. Expression of ORFs or shRNAs was induced by addition of 0.5 μg/ml doxycycline to cell culture media for 7-10 days. pLUT expressing turbo red fluorescent protein (RFP) and pGIPZ/TRIPZ expressing non-targeting shRNA were used as appropriate controls.

Addison J.B., Voronkova M.A., Fugett J.H., Lin C.C., Linville N.C., Trinh B., Livengood R.H., Smolkin M.B., Schaller M.D., Ruppert J.M., Pugacheva E.N., Creighton C.J, & Ivanov A.V. (2021). Functional hierarchy and cooperation of EMT master transcription factors in breast cancer metastasis. Molecular cancer research : MCR, 19(5), 784-798.

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