Plenti c myc ddk ires neo vector

Manufactured by OriGene

The PLenti-C-Myc-DDK-IRES-Neo vector is a lentiviral expression vector that allows for the expression of a gene of interest along with a Myc-DDK tag and a neomycin resistance marker. The vector utilizes the CMV promoter to drive gene expression. It is designed for the generation of stable cell lines expressing the gene of interest.

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Lab products found in correlation

Platinum pfx polymerase, by Thermo Fisher Scientific (1 mentions) Interleukin 3 (il 3), by Merck Group (1 mentions) Virapower lentiviral packaging mix, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

PLenti-C-Myc-DDK vector (6 citations) PLenti vector (4 citations) PLenti-C-Myc-DDK-IRES-Neo (2 citations) C-Myc (2 citations)

3 protocols using plenti c myc ddk ires neo vector

HILPDA Truncation Mutant Generation

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Human HILPDA truncation mutants were subcloned from the pcDNA3.1+/C-(K)DYK(HsHILPDA) ORF clone (Genscript OHu08913). Primers with a 5’ Sgf1 (forward) and 3’ Mlu1 (reverse) linker sequences (restriction sites underlined) were used to amplify truncated coding sequences by PCR (Platinum PFX polymerase, Life Technologies 11708013) under the following conditions: denaturation 15 seconds at 94°, annealing 30 seconds at 55°, and extension 1 minute at 68° for a total of 35 cycles. PCR products were purified, digested and ligated into the pLenti-C-Myc-DDK-IRES-Neo vector (Origene, PS100081) and confirmed by sequencing. The primer sequences used are: HsHIG2-For 5-ATATAGCGATCGCCATGAAGCATGTGTTGAACCTC-3, Rev64 5-ATATAACGCGTCATGCTTCTGGATGGATGGTC-3, Rev37 5-ATATAACGCGTCCCAGGCGATGGGCTCTCTAG-3, Rev34 5-ATATAACGCGTTGGGCTCTCTAGTAAGCCCTC-3, Rev31 5-ATATAACGCGTTAGTAAGCCCTCTAGGGACTCC-3, Rev28 5-ATATAACGCGTCTCTAGGGACTCCATCACTCTAAC-3. The double nickase CRISPR plasmid set targeting human HILPDA was from Santa Cruz (sc-403510-NIC)

VandeKopple M.J., Wu J., Auer E.N., Giaccia A.J., Denko N.C, & Papandreou I. (2019). HILPDA regulates lipid metabolism, lipid droplet abundance, and response to microenvironmental stress in solid tumors.. Molecular cancer research : MCR, 17(10), 2089-2101.

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Lentiviral Transduction of PEX13 Mutants

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pMXs‐IP‐HA‐Parkin 20 (Addgene #38248) was cotransfected with the helper plasmids pUMVC and pCMV‐VSV‐G 42 (Addgene #8849 and #8454) into HEK293T cells. PEX13 cDNAs containing WT, W313G mutation, and I326T mutation were cloned into pLenti‐C‐Myc‐DDK‐IRES‐Neo vector (Origene), then cotransfected into HEK293 cells with the helper plasmids pCMVΔR8.91 43 and pMDG 44. Retro‐ or lentiviral supernatant was filtered through a 0.45‐μm membrane and then added to target cells in the presence of polybrene (8 μg/ml). Cells were selected in media containing 0.5 μg/ml puromycin or 500 μg/ml G418 and then maintained in media containing 0.25 μg/ml puromycin and/or 100 μg/ml G418.

Lee M.Y., Sumpter R J.r., Zou Z., Sirasanagandla S., Wei Y., Mishra P., Rosewich H., Crane D.I, & Levine B. (2016). Peroxisomal protein PEX13 functions in selective autophagy. EMBO Reports, 18(1), 48-60.

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Engineered Ba/F3 Cell Lines for PDGFRA Mutations

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Ba/F3 cells were grown in RPMI-1640 medium with 10% FBS at 37°C and 5% CO 2 . Lentiviruses were produced by transient transfection of HEK-293T cells with ViraPower Lentiviral Packaging Mix (Invitrogen, #K497500) plus the pLENTI-C-Myc-DDK-IRES-Neo vector (Origene) containing full-length PDGFRA cDNA encoding each of the described mutations. Ba/F3 cells were then transduced with filtered lentivirus and polybrene via spinoculation and supplemented with murine IL3 (Cell Signaling Technology, #8923C). Transduced, IL3-dependent Ba/ F3 cells were then selected for 1 week in G418 (Sigma, #A1720). In order to generate IL3-independent clones, G418-resistant Ba/F3 cells were plated in limiting dilutions in the absence of IL3 and grown for 1 to 3 weeks (48) . Full sequencing of the PDGFRA cDNA was performed for each clonal IL3-independent Ba/F3 cell line. The PDGFRA mutation P653_H654insQGP is abbreviated as QGPins in the text and figures.

Grunewald S., Klug L.R., Mühlenberg T., Lategahn J., Falkenhorst J., Town A., Ehrt C., Wardelmann E., Hartmann W., Schildhaus H.U., Treckmann J., Fletcher J.A., Jung S., Czodrowski P., Miller S., Schmidt-Kittler O., Rauh D., Heinrich M.C, & Bauer S. (2021). Resistance to Avapritinib in PDGFRA-Driven GIST Is Caused by Secondary Mutations in the PDGFRA Kinase Domain. Cancer discovery, 11(1).

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