Pcmv neo 6 vector

Manufactured by OriGene

Sourced in United States

The PCMV-Neo-6 vector is a plasmid that contains a neomycin resistance gene under the control of the cytomegalovirus (CMV) promoter. This vector can be used for the selection and maintenance of stably transfected mammalian cell lines.

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PCMV vector (6 citations)

2 protocols using pcmv neo 6 vector

Construction and Validation of EGFR Plasmids

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A human wild-type EGFR expression plasmid (pCMV-E1wt) was constructed. The coding sequence of EGFR (a gift from Dr S. Yokoyama, RIKEN) [37 (link)] was amplified by PCR. The PCR product was introduced into an EcoRV-XbaI restriction site in the pCMV-Neo-6 vector (OriGene, Rockville, MD, USA). A plasmid containing the EGFR-6KR mutant was constructed as described previously [13 (link)]. With the plasmid as a template, an EGFR-6KR gene was cloned into pCMV-Neo-6 vector using the same primers indicated in the pCMV-E1wt section. The resulting plasmid was named pCMV-E1-6KR. MCF-7 cell lines over-expressing receptors were established by transfecting with either the plasmids reported above or the pCMV-Neo-6 vector as a control cell line. Stable receptor clones containing those plasmids were selected and maintained by treating with 200 μg·mL⁻¹ G418. For selection of the clones, EGFR expression and phosphorylation of EGFR and ERK in response to growth factor stimulation were examined by western blotting.

Nagashima T., Inoue N., Yumoto N., Saeki Y., Magi S., Volinsky N., Sorkin A., Kholodenko B.N, & Okada-Hatakeyama M. (2015). Feedforward regulation of mRNA stability by prolonged extracellular signal-regulated kinase activity. The Febs Journal, 282(4), 613-629.

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EGFR Expression Plasmid Construction

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A human wild type EGFR expression plasmid (pCMV-E1wt) was constructed as follows. The coding sequence of EGFR (a gift from Dr. Shigeyuki Yokoyama, RIKEN) [37 (link)] was amplified by PCR. The PCR product was introduced into an Eco RV-Xba I restriction site in the pCMV-Neo-6 vector (OriGene, Rockville, MD). A plasmid containing the EGFR-6KR mutant was constructed as described earlier [13 (link)]. With the plasmid as a template, an EGFR-6KR gene was cloned into pCMV-Neo-6 vector using the same primers indicated in the pCMV-E1wt section. The resulting plasmid was named pCMV-E1-6KR. MCF-7 cell lines over-expressing receptors were established by transfecting with either the plasmids shown above or the pCMV-Neo-6 vector as a control cell line. Stable receptor clones containing those plasmids were selected and maintained by treating with 200 µg/mL G418. For selection of the clones, EGFR expression and phosphorylation of EGFR and ERK in response to growth factor stimulation were examined by western blot.

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