Plox ttag irestk

Manufactured by Addgene

The PLOX-Ttag-iresTK is a lab equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

Automatically generated - may contain errors

Lab products found in correlation

Schneider s medium, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Effectene, by Qiagen (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Metafectene, by Biontex (2 mentions) Tc20 automated cell counter, by Bio-Rad (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Laminin, by Merck Group (1 mentions) Polybrene, by Merck Group (1 mentions) Galactose, by Merck Group (1 mentions) Cct020312, by Merck Group (1 mentions) Gsk2606414, by Merck Group (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Sodium pyruvate, by Merck Group (1 mentions) Mtor inhibitor rapamycin, by LC Laboratories (1 mentions) Penicillin streptomycin, by Lonza (1 mentions) Pcr2.1 ta cloning vector, by Thermo Fisher Scientific (1 mentions) Trizol plus rna purification system, by Thermo Fisher Scientific (1 mentions) Pwpxld, by Addgene (1 mentions)

7 protocols using plox ttag irestk

Isolation and Characterization of Fancd2-/- Mouse Cells

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Fancd2^-/- mice (Fancd2^tm1Hou, MGI code: 2673422, backcrossed into C57BL/6 background for at least 11 generations), obtained from K.J. Patel [42 (link), 43 (link)], were maintained in a conventional mouse facility. All animal experiments undertaken in this study were performed under the approval of the EU Directive 2010/63EU, Spanish law RD53/2013 and the Hospital Virgen del Rocio Ethical Review Committee. Timed matings between Fancd2^+/− males and females were set up. Females were checked for the presence of a vaginal plug the following morning, and considered to be at day E0.5 of pregnancy. Pregnant females were sacrificed at E13.5, uteruses removed and embryos dissected. Murine embryonic fibroblasts (MEFs) cultures were obtained, genotyped and transformed using a lentiviral vector pLOX-Ttag-iresTK (addgene 12246). Clones were isolated and expanded.
Murine bone marrow cells from femora and tibias were obtained by flushing in 2 mls of PBS+3% fetal bovine serum. Cells were enumerated using trypan blue 0.2% in a TC20 Automated Cell Counter (Bio-Rad). Biotinylated B220 (cloneRA3-6B2), Gr1 (Clone RB6-8C5) were obtained from BDBioscience. Cells were enriched using Streptavidin-bound magnetic particles (BD IMag) according to manufacturer instructions.

García-Rubio M.L., Pérez-Calero C., Barroso S.I., Tumini E., Herrera-Moyano E., Rosado I.V, & Aguilera A. (2015). The Fanconi Anemia Pathway Protects Genome Integrity from R-loops. PLoS Genetics, 11(11), e1005674.

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Fibroblast and HeLa Cell Culture Protocols

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Fibroblasts were derived from patient skin and HeLa cells were obtained from ATCC (CCL-2), which were grown in high-glucose or galactose DMEM (Dulbecco’s Modified Eagle Medium) supplemented with Glutamax, sodium pyruvate, 10% FBS (fetal bovine serum), penicillin/streptomycin (Gibco Life Technologies) in humidified atmosphere at 37°C and 5% CO₂. Immortalisation and cell rescue were performed by lentiviral infection (pLOX-Ttag-iresTK, Addgene plasmid #12246, pWPXLd-APOO-HA and pWPXLd-Ires-Puro^R ‘empty vector’). Transfections were performed using Metafectene (Biontex).
S2R+ cells were grown at 25°C in Schneider’s medium (Thermo Fisher) with 10% FBS (Gibco). S2R+ cells were transfected using Effectene (Qiagen).

Benincá C., Zanette V., Brischigliaro M., Johnson M., Reyes A., do Valle D.A., J. Robinson A., Degiorgi A., Yeates A., Telles B.A., Prudent J., Baruffini E., S. F. Santos M.L., R. de Souza R.L., Fernandez-Vizarra E., J. Whitworth A, & Zeviani M. (2020). Mutation in the MICOS subunit gene APOO (MIC26) associated with an X-linked recessive mitochondrial myopathy, lactic acidosis, cognitive impairment and autistic features. Journal of Medical Genetics, 58(3), 155-167.

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Fibroblast Immortalization and Rescue

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Fibroblasts were derived from the patient's skin biopsy and grown (high-glucose Dulbecco's Modified Eagle Medium Glutamax, sodium pyruvate, 10% fetal bovine serum, and penicillin/streptomycin) at 37°C (5% CO₂). Cell immortalization and rescue experiments were performed by lentiviral transduction (pLOX-Ttag-iresTK, Addgene #12246, pWPXLd-POLR3A-HA “WT or V1241M”and “empty vector”).
For Western blot, cells were lysed (Tris-HCl 20 mM, NaCl 150 mM, EDTA 1 mM, Triton-X-100 1%, glycerol 10%, and MgCl₂ 1.5 mM plus protease inhibitors), centrifuged, and supernatants mixed with loading buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Protein concentration was measured (DC protein assay; Bio-Rad).

Zanette V., Reyes A., Johnson M., do Valle D., Robinson A.J., Monteiro V., Telles B.A., L.R. Souza R., S.F. Santos M.L., Benincá C, & Zeviani M. (2020). Neurodevelopmental regression, severe generalized dystonia, and metabolic acidosis caused by POLR3A mutations. Neurology: Genetics, 6(6), e521.

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Lentiviral Transduction of Porcine Stem Cells

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Two lentiviral vectors, namely pLOX-Ttag-iresTK (addgene No. 12246) and Ef1a-Large T-Ires-Puro (addgene No. 18922), were purchased from addgene. Lentiviruses were produced by co-transfecting the either transfer vector and the 2^nd generation packaging vectors psPAX2 and pMD2.G into HEK293T cells, and were concentrated using ultracentrifugation. The porcine cells collected after differential plating were seeded to 12-well plates coated with laminin (20 μg/mL; Sigma-Aldrich), and transduced with the prepared lentiviruses by using “spinfection”. This method can be used to transduce SSCs with substantial efficiency [22 (link)]. Briefly, the cells exposed to 10 μg/mL polybrene (Sigma-Aldrich) and the concentrated virus supernatant at a multiplicity of infection (MOI) of 100 were centrifuged at 3,000×g for 1 h at 32 °C, followed by 16 h of incubation at 37 °C. One day after lentiviral transduction, cells were subjected to FACS employing an antibody against PLD6 (rabbit anti-PLD6; ab237612, Abcam). The sorted PLD6⁺ cells were seeded to 24-well plates coated with laminin (20 μg/mL; Sigma-Aldrich) for expansion.

Zheng Y., Feng T., Zhang P., Lei P., Li F, & Zeng W. (2020). Establishment of cell lines with porcine spermatogonial stem cell properties. Journal of Animal Science and Biotechnology, 11, 33.

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Immortalized Fibroblasts for mtDNA Segregation

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Fibroblasts from homoplasmic and heteroplasmic mice were isolated from mouse ear and immortalized by transfection with pLOX-Ttag-iresTK (Addgene). Cell lines were grown in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 5% FBS, 1% penicillin-streptomycin (Lonza), and 1 mM sodium pyruvate (Sigma-Aldrich). Where indicated, the carbon source was 5 mM glucose (Sigma-Aldrich) or 5 mM galactose (Sigma-Aldrich) or Albumax lipid-rich bovine serum albumin (BSA; 1 mg/ml). For in vitro assessment of mtDNA segregation using modulators of the autophagy flux, we used 1 μM final concentration of the uncoupling agent CCCP (Sigma-Aldrich), mTOR inhibitor rapamycin (LC Laboratories), PERK activator (CCT020312, Millipore), and PERK inhibitor (GSK2606414, Millipore).

Lechuga-Vieco A.V., Latorre-Pellicer A., Johnston I.G., Prota G., Gileadi U., Justo-Méndez R., Acín-Pérez R., Martínez-de-Mena R., Fernández-Toro J.M., Jimenez-Blasco D., Mora A., Nicolás-Ávila J.A., Santiago D.J., Priori S.G., Bolaños J.P., Sabio G., Criado L.M., Ruíz-Cabello J., Cerundolo V., Jones N.S, & Enríquez J.A. (2020). Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics. Science Advances, 6(31), eaba5345.

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Fibroblast and HeLa Cell Culture Protocol

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Fibroblasts were derived from patient skin and HeLa cells were obtained from ATCC (CCL-2), which were grown in high-glucose or galactose DMEM (Dulbecco's Modified Eagle Medium) supplemented with Glutamax, sodium pyruvate, 10% FBS (fetal bovine serum), penicillin/streptomycin (Gibco Life Technologies) in humidified atmosphere at 37°C and 5% CO₂. Immortalisation and cell rescue were performed by lentiviral infection (pLOX-Ttag-iresTK, Addgene plasmid #12246, pWPXLd-APOO-HA and pWPXLd-Ires-Puro^R ‘empty vector’). Transfections were performed using Metafectene (Biontex).
S2R+ cells were grown at 25°C in Schneider’s medium (Thermo Fisher) with 10% FBS (Gibco). S2R+ cells were transfected using Effectene (Qiagen).

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Amplification and Cloning of APOPT1 cDNA

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For the amplification of APOPT1 cDNA, total RNA was extracted from HeLa and HEK293T cells using the TRIzol Plus RNA Purification System (Invitrogen‐ThermoFisher Scientific) and retrotranscribed with the Omniscript RT kit (Qiagen). Approximately 200 ng of cDNA was used as template for the amplification of APOPT1 using specific primers (Table 1). C‐terminal HA tags were added by PCR amplification, and the GFP tag was added by cloning a stop codon‐less APOPT1 cDNA in frame with EGFP already inserted into pCR2.1. The PCR generated fragments in pCR2.1 TA cloning vector (Invitrogen) were then cloned into pWPXLd‐ires‐Puro^R and pWPXLd‐ires‐Hygro^R lentiviral expression vectors, modified versions of pWPXLd (Addgene #12258), by restriction enzyme digestion with PmeI and BamHI and ligation using T4 DNA ligase.
Lentiviral particles were generated in HEK293T packaging cells by co‐transfection of the target vector with the packaging psPAX2 (Addgene plasmid #12260) and envelope pMD2.G (Addgene #12259) vectors. Target cells were transduced as described (Perales‐Clemente et al, 2008). Twenty‐four hours after transduction, cells were selected for puromycin or hygromycin resistance.
Primary skin fibroblasts were immortalized by lentiviral transduction of pLOXTtag‐iresTK (Addgene #12246).
All the utilized lentiviral vectors were a gift from Didier Trono.

Signes A., Cerutti R., Dickson A.S., Benincá C., Hinchy E.C., Ghezzi D., Carrozzo R., Bertini E., Murphy M.P., Nathan J.A., Viscomi C., Fernandez‐Vizarra E, & Zeviani M. (2018). APOPT1/COA8 assists COX assembly and is oppositely regulated by UPS and ROS. EMBO Molecular Medicine, 11(1), e9582.

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