Pmxs puro vector

Manufactured by Cell Biolabs

Sourced in United States

The PMXs-puro vector is a lentiviral expression vector that contains a puromycin resistance gene. It can be used for stable gene expression in mammalian cells.

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Lab products found in correlation

Gblock gene fragment, by Integrated DNA Technologies (1 mentions) Topo ta, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PMXs-miR-GFP/puro retroviral expression vector PMXs-IRES-Puro vector PMXs-puro retroviral vector N-MYC-tagged pMXs-Puro Retroviral Vector PMXs-puro

3 protocols using pmxs puro vector

Retroviral Transduction of CYLD Mutants

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The CYLD wild-type (WT), and various CYLD mutants were generated by gene synthesis and cloned into the pMXs-puro vector (Cell Biolabs, USA). The pMXs-puro-EGFP plasmid was used as control. The retroviral packaging cell line, Plat A cells were used to generate infective retroviruses for C666-1 and HK1-EBV infection according to the manufacturer's instructions. Seven days after infection, cells were collected for the confirmation of CYLD mutant expression by immunoblotting. For growth assay, cells were plated at low density (1 × 10⁴ per well) in RPMI (for C666-1) or DMEM (for HK1-EBV) with 4% FBS and assessed for proliferation at 48 h. Mutant-induced cell growth, compared with the EGFP vector control or CYLD WT, was analysed by MTT assay. C666-1 and HK1-EBV cell lines were previously established and authenticated in our laboratory. No mycoplasma contamination was detected in these cell lines. An uncropped images of western blot membranes for the expression of CYLD WT and mutant protein is shown in Supplementary Fig. 15.

Li Y.Y., Chung G.T., Lui V.W., To K.F., Ma B.B., Chow C., Woo J.K., Yip K.Y., Seo J., Hui E.P., Mak M.K., Rusan M., Chau N.G., Or Y.Y., Law M.H., Law P.P., Liu Z.W., Ngan H.L., Hau P.M., Verhoeft K.R., Poon P.H., Yoo S.K., Shin J.Y., Lee S.D., Lun S.W., Jia L., Chan A.W., Chan J.Y., Lai P.B., Fung C.Y., Hung S.T., Wang L., Chang A.M., Chiosea S.I., Hedberg M.L., Tsao S.W., van Hasselt A.C., Chan A.T., Grandis J.R., Hammerman P.S, & Lo K.W. (2017). Exome and genome sequencing of nasopharynx cancer identifies NF-κB pathway activating mutations. Nature Communications, 8, 14121.

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Generating Retroviral and Lentiviral Vectors

Cited 1 time

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pMXs-puro vector (Cell Biolabs) was equipped with a novel multiple cloning site with or without N- or C-terminal tags (RFP, GFP and FLAG). SPPL3 and its inactive mutant SPPL3 D271A (kind gift from Dr. R. Flührer) (Voss et al., 2012 (link)) were recloned into pMXs-puro-RFP or -GFP using XhoI/BamHI restriction sites. B3GNT5 and B4GALNT1 were PCR amplified from IMAGE:202800754 and IMAGE:202800771 and cloned into pMXs-puro-FLAG-C or -N by EcoRI/BamHI restriction sites. ST3GAL5 was amplified from IMAGE:202759803 and cloned into pMXs-puro-FLAG-C using EcoRI/BclI digestion into an EcoRI/BamHI digested plasmid. The pLZRS-based retroviral vectors containing HLA-A*02:01/B*40:01/C*03:03-IRES-ΔNGFR were described before (Griffioen et al., 2012 (link); Van Bergen et al., 2010 (link)). Generation of retroviral supernatants and transduction of cells were performed as described (Spaapen et al., 2008 (link)). HLA-C*05:01 (IMGT/HLA database) with a mutated signal peptide from HLA-A*02:01 (M4V) was purchased as a gBlock gene fragment (Integrated DNA Technologies, Inc.) and used as a template for amplification. The PCR product was cloned into the puc2CL6IN lentiviral vector using NheI and BamHI. gRNAs (Table S4) were cloned into the pX330 expression vector or the lentiviral vectors lentiCRISPR_v2 or pLCRISPR.efs.GFP (Addgene) as described (Heckl et al., 2014 (link); Sanjana et al., 2014 (link)).

Jongsma M.L., de Waard A.A., Raaben M., Zhang T., Cabukusta B., Platzer R., Blomen V.A., Xagara A., Verkerk T., Bliss S., Kong X., Gerke C., Janssen L., Stickel E., Holst S., Plomp R., Mulder A., Ferrone S., Claas F.H., Heemskerk M.H., Griffioen M., Halenius A., Overkleeft H., Huppa J.B., Wuhrer M., Brummelkamp T.R., Neefjes J, & Spaapen R.M. (2020). The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses. Immunity, 54(1), 132-150.e9.

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Retroviral Vectors for JAK3 Mutation Screening

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Retroviral vectors harboring novel JAK3 mutations were generated through site-directed mutagenesis and were introduced into Ba/F3 and NIH-3T3 cells to test transformation capacity. The coding region of JAK3 (NM_000215.3) was amplified and cloned into vector TOPO TA (Invitrogen, Grand Island, NY). Mutations of JAK3 with 1847 T > C and 1866 A > G in the coding region were generated by site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) with mutation-specific primers according to the manufacturer's instructions. The Platinum-E retroviral packaging cell line was seeded in Dulbecco's modified Eagle's medium with 10% (v/v) fetal bovine serum. The following day, the cells were transfected with the JAK3 mutant construct in the pMXs-Puro vector (Cellbio Labs, San Diego, CA) and the packaging plasmids pCL-eco using Fugene6 transfection reagents (Roche, Indianapolis, IN). Viral supernatants were collected 48 to 72 hours later. Ba/F3 and NIH-3T3 cells in 6-well plates or 10-cm dishes were inoculated with 1 mL of viral supernatant in 1 mL of medium with polybrene (4 mg/mL). The transduced cells were selected using puromycin.

Sim S.H., Kim S., Kim T.M., Jeon Y.K., Nam S.J., Ahn Y.O., Keam B., Park H.H., Kim D.W., Kim C.W, & Heo D.S. (2017). Novel JAK3-Activating Mutations in Extranodal NK/T-Cell Lymphoma, Nasal Type. The American journal of pathology, 187(5).

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