Human HER2 cDNA (RC212583; Origene) was subcloned into pMXs-IRES-Blasticidin (Cell Biolabs). HER2 mutants were generated by site-directed mutagenesis. Retroviral expression vector retrovirus was produced by transient transfection of HEK 293T cells with the retroviral ERBB expression vector pMXs-IRES-Blasticidin, pCMV-Gag-Pol vector, and pCMV-VSV-G-Envelope vector (Cell Biolabs). Briefly, HEK 293T cells were plated (2.5 × 105 per plate) in 6-well plates (Corning) and incubated overnight. The next day, retroviral plasmids (1 µg of ERBB, 0.32 µg of pCMV-Gag-Pol, and 0.16 µg pCMV-VSV-G) were mixed in 150 µL of Opti-MEM (Life Technologies). The mixture was incubated at room temperature for 5 minutes and then added to Opti-MEM containing transfection reagent Lipofectamine (Invitrogen) and incubated for 20 minutes. Mixture was then added dropwise to HEK 293T cells. The next day, the medium was replaced with fresh culture medium, and retrovirus was harvested at 24 hours.
Pmxs ires blasticidin
Manufactured by Cell Biolabs
The PMXs-IRES-Blasticidin is a mammalian expression vector that allows for the coupled expression of a gene of interest and the blasticidin resistance gene. The internal ribosome entry site (IRES) enables the translation of both the gene of interest and the blasticidin resistance gene from a single mRNA transcript.
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Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Pcmv vsv g envelope vector, by Cell Biolabs (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
6 well plate, by Corning (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Pbabe k ras 12v, by Addgene (1 mentions)
4 protocols using pmxs ires blasticidin
1
Retroviral Expression of HER2 Mutants
2
Cell Lines for Cytoskeletal Imaging
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Plasmids coding for mCherry-α-tubulin and mCardinal-ZO1-C-14 were PCR-amplified from pmCherry_α_tubulin_IRES_puro2 (kindly provided by Daniel Gerlich) [67 (link)] and from mCardinal-ZO-1-C-14 (gift from Michael Davidson; Addgene plasmid # 56179), respectively, and then ligated into pMXs-IRES-Blasticidin (Cell Biolabs Inc.). Doxycycline-inducible MDCK II cells were generated using the same two-step transduction strategy as described previously [21 (link)] and enriched by antibiotic selection using hygromycin (Life Technologies) at 1600 µg ml−1 and puromycin (Sigma-Aldrich) at 2 µg ml−1. Inducible MDCK II cells stably expressing mCherry-α-tubulin or mCardinal-ZO-1 were generated by retroviral transduction and subsequent selection in 5 µg ml−1 blasticidine (Novus Biologicals). GFP-CEP131 was PCR-amplified from pEGFP-C2-CEP131 plasmid [68 (link)] and ligated into pRetroX-Tight-Puro using Not1 and Mlu1 cloning sites. Expression of transgenes coding for EGFP-tagged centrosomal proteins was induced by treatment of cells with 2.5 µg ml−1 of Doxycycline (Sigma-Aldrich) for 24 or 48 h, as indicated.
3
Generating Protein Tagging Vectors
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To generate pCMV enhancer vectors, the pCDNA3.1 (Invitrogen) backbone was used. For the MoMuLV enhancer vectors, pMXs-IRES-Blasticidin or pMXs-IRES-Puromycin (Cell Biolabs) were used. The BirA ORF was PCR amplified from the vector pBBHN (34 (link)). The BAP domain fragments were prepared by designing oligonucleotides and sequential PCR, followed by restriction digestion and insertion into the pCDNA3.1 or pMXs-IRES-Puromycin vector. The insert sequences were confirmed by sequencing. The vectors generated were used to clone the ORFs Emerin, GFP, H2A, H2A.Z, H3.1 and macroH2A1.
4
Doxycycline-Inducible Cell Line Generation
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cDNA encoding mCherry‐α‐tubulin, mCardinal‐H1, and dTomato‐VE‐cadherin were PCR‐amplified from pmCherry_α_tubulin_IRES_puro2 [kindly provided by Daniel Gerlich (Steigemann et al, 2009 )], mCardinal‐H1‐10 [a gift from Michael Davidson (Chu et al, 2014 ; Addgene plasmid # 56161)], and tdTomato‐VE‐cadherin‐N‐10 (a gift from Michael Davidson (Addgene plasmid # 58142), respectively, and subsequently ligated into pMXs‐IRES‐Blasticidin (Cell Biolabs Inc.).
MCF10A ecoR cell lines allowing doxycycline‐inducible expression of EGFP‐NLP, EGFP‐CEP68 and EGFP‐PLK4 were described previously (Schnerch & Nigg,2016 ). Doxycycline‐inducible MDCK II cells were generated using the same two‐step transduction strategy and enriched by antibiotic selection using hygromycin at 400 μg ml−1 and puromycin at 2 μg ml−1. Inducible MDCK and MCF10A cells stably expressing mCherry‐α‐tubulin, dTomato‐E‐cadherin and mCardinal H1 were generated by retroviral transduction and sorted by flow cytometry using BD FACSAria IIIu cell sorter (FACS Core Facility of Biozentrum). MCF10A ecoR cells stably expressing K‐Ras were obtained by ecotropic retroviral transduction with pBabe K‐Ras 12V [Addgene plasmid #12544, gift from Channing Der (Khosravi‐Far et al, 1996 )].
MCF10A ecoR cell lines allowing doxycycline‐inducible expression of EGFP‐NLP, EGFP‐CEP68 and EGFP‐PLK4 were described previously (Schnerch & Nigg,
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