Pci neo vector

Manufactured by Promega

Sourced in United States

The PCI-neo vector is a laboratory plasmid vector used for DNA cloning and expression. It contains a neomycin resistance gene for selection of transformed cells and a multiple cloning site for insertion of DNA sequences.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions) Pci neo expression vector, by Promega (2 mentions) Dcas9 vp64 p65 rta construct19, by Addgene (2 mentions) Sc 423402 act, by Santa Cruz Biotechnology (2 mentions) Anti ha hrp, by Merck Group (2 mentions) Anti ha beads, by Merck Group (2 mentions) Anti yap antibody d8h1x, by Cell Signaling Technology (2 mentions) Clone 4a6 antibody, by Merck Group (2 mentions) Turbofect, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b, by GE Healthcare (2 mentions) Ni nta agarose, by Qiagen (2 mentions) Mg132, by Merck Group (1 mentions) Pgem4z, by Promega (1 mentions) Penicillin, by Sartorius (1 mentions) Streptomycin, by Sartorius (1 mentions) L glutamine, by Sartorius (1 mentions) Fetal bovine serum (fbs), by Sartorius (1 mentions) Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions) Transit lt1 reagent, by Geneflow (1 mentions)

55 protocols using pci neo vector

Generation of Bcl-2 Family Protein Constructs

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Full-length cDNAs of human RNF183, Bcl-xL, and other Bcl-2 family members (Bcl-2s: Bcl-2, BAX, and BAD) were amplified from the cDNA library of HeLa cells or 293T cells by RT-PCR. The PCR product of wild-type or mutated RNF183/Bcl-xL/Bcl-2s was digested by EcoRI and SalI and subsequently cloned into a series of pCI-neo vectors (Promega) constructed with the following tags generated by our laboratory: Flag (Flag epitope at N or C terminus), 2xHA (HA epitope at C terminus), and GFP (GFP at N terminus).
Wild-type Ub and its lysine to arginine mutants (K29R, K48R, and K63R) were all constructed with pCI-neo vector via EcoRI and XbaI sites harboring Myc tag or HA tag.
GST-fusion RNF183 and its mutant were constructed by inserting a Myc tag at the N terminus of RNF183 into pGEX-6P-1. Bcl-2 and Bcl-xL were also constructed into pGEX-6P-1, but with an HA tag at the N terminus. Ub, UBA1, and UBCU5C expression plasmids were a kind gift from Ronggui Hu, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS, Shanghai, China. Proteins were purified under native conditions using glutathione-Sepharose 4B (GE Healthcare Life Sciences) or Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions. All constructs were verified by sequencing.

Wu Y., Li X., Jia J., Zhang Y., Li J., Zhu Z., Wang H., Tang J, & Hu J. (2018). Transmembrane E3 ligase RNF183 mediates ER stress-induced apoptosis by degrading Bcl-xL. Proceedings of the National Academy of Sciences of the United States of America, 115(12), E2762-E2771.

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Calcium Signaling in HEK293T Cells

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The cDNA encoding GCaMP6 was amplified from HBT-GCaMP6-HA43 (Liu et al., 2017) , and inserted into pCI-neo vector (Promega). The cDNAs encoding CNGC8, CNGC18, and CAM2 were cloned into pCI-neo vector (Promega), respectively. In one case, CNGC18 and CAM2 were cloned into a single vector, pBudCE4.1 (Invitrogen), for co-expression. HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37 C with 5% CO2. Transfections were performed using a Lipofectamine 3000 Transfection Reagent Kit (Invitrogen). Plasmid DNA of each construct containing CNGC8, CNGC18, CAM2, or the combination of these constructs, was co-transfected with the plasmid containing GCaMP6 at a 10:1 ratio. HEK293T cells showing green fluorescence indicating GCAMP expression were used for time lapse Ca 2+ imaging experiments. HEK293T cells expressing GCaMP6 were monitored by a Zeiss AxioObserver Z1 Inverted Microscope using a 320 objective. The interval of data acquisition was 5 seconds. The software Ivision4.5 (BioVision Technologies) was used for data acquisition and analysis. The working solution for Ca 2+ imaging contained (in mM) 120 NaCl, 3 KCl, 1 MgCl 2 , 1.2 NaHCO 3 , 10 Glucose, 10 Hepes, pH 7.2. About 60 seconds after initiation of imaging procedure, 2.5 mM CaCl 2 was added to elicit Ca 2+ entry through any active channels.

Pan Y., Chai X., Gao Q., Zhou L., Zhang S., Li L, & Luan S. (2019). Dynamic Interactions of Plant CNGC Subunits and Calmodulins Drive Oscillatory Ca²⁺ Channel Activities. Developmental cell, 48(5).

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CD47 Antibody Engineering and Characterization

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Example 2

ELISA positive binders with OD₄₅₀>0.5 were selected for sequencing analysis. Heavy chains of identified CD47 binders were engineered onto a human IgG4 scaffold to minimize recruitment of Fc-dependent effector functions such as ADCC and CDC. The amino acid sequences of the light chain and heavy chain variable (Vh) region, and the reformatted full length heavy chain sequences (in IgG4 isotype) of identified CD47 binders were listed in Table 1 and Table 2. Individual reformatted CD47 antibody heavy chain and corresponding light chain were then sub-cloned onto separate pCI-neo vector (Promega, USA) for pairwise HEK293 transfection, expression, purification, and characterization.

US11390677B2. Human anti-CD47 antibodies and uses thereof (2022-07-19). Trican Biotechnology Co., Ltd [TW]. Inventors: Huang-Tsu Chen [US], Jiun-Shyang Leou [TW], Chung-Yuan Hsu [TW], Cheng-Ke Li [TW], Yun-Ting Wang [TW], Li-Tsen Lin [TW], Shiou-Ting Chen [TW], Chen-Wei Chiang [TW].

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Transfection and treatment of HEK293T cells

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Human embryonic kidney 293T (HEK293T) cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal calf serum at 37 °C in 5% CO₂ infusion and humidified air. 293T cells were transfected with plasmids by Lipofectamine 2000 (Invitrogen). Human full-length MEN1 complementary DNA was inserted in pCI-neo vector (Promega) to create the human MEN1 expression construct. β-catenin plasmid with green fluorescent protein tag was obtained from Xiang Yu (Institute of Neuroscience, SIBS, CAS) with permission from James Nelson (Stanford University). MG132 (25 μM, Sigma) was used to treat transfected 293T cells for 2 h before harvest.

Jiang X., Cao Y., Li F., Su Y., Li Y., Peng Y., Cheng Y., Zhang C., Wang W, & Ning G. (2014). Targeting β-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours. Nature Communications, 5, 5809.

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Cloning and Expression of Human DDT

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All SOD1 plasmids, including pCI-hSOD1^WT and pCI-hSOD1^G93^A were generated previously by our group, by inserting human SOD1 constructs into the pCI-NEO vector (Promega), between the EcoRI and the NotI sites. A DDT expressing plasmid pET-21b-hDDT, was generously received from the Bernhagen group (Munich, Germany). The sequence of human DDT was transferred from the pET-21b vector^{75 (link)} into the mammalian expression vector pcDNA3.1 (-) vector, between the BamHI and NotI restriction sites. Human DDT gene was amplified by PCR from pET-21b vector using following primers:
Forward primer: 5’-ATTACGCGGCCGCCACCATGCCGTTCCTGGAG-3’.
Reversed primer: 5’-GCTAGGATCCCTATAAAAAAGTCATGAC-3’.

Alaskarov A., Barel S., Bakavayev S., Kahn J, & Israelson A. (2022). MIF homolog d-dopachrome tautomerase (D-DT/MIF-2) does not inhibit accumulation and toxicity of misfolded SOD1. Scientific Reports, 12, 9570.

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Mammalian Cell Protein Expression Protocols

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Constructs for protein expression in mammalian cell culture were inserted into the pCI-neo vector (Promega) with an N-terminal FLAG (DYKDDDDK) tag. Templates for in vitro transcription of radiolabeled mRNA were EcoRI/BamHI cloned into pGEM-4Z (Promega). MINX, MINX Δi and the expression vectors for FLAG-Y14, FLAG-eIF4A3, FLAG-CWC22 and their respective mutants were described previously (20 (link),24 (link)). CWC22 deletion mutants 110–908, 340–908, 1–769 and Δ669–759 were polymerase chain reaction (PCR) amplified and inserted into pCI-neo-FLAG. AdML-PT60 and AdML-PT60/e1(18) were generated by PCR amplification of a synthetic DNA. All constructs were verified by DNA sequencing.

Steckelberg A.L., Altmueller J., Dieterich C, & Gehring N.H. (2015). CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes. Nucleic Acids Research, 43(9), 4687-4700.

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Synthetic Transcriptional Activators for CRISPR Modulation

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VP64 and VP160 sequences containing 4 or 10 copies of the nucleotides encoding VP16 minimal transactivator sequence DALDDFDLDML^{18 (link)} (Table S3) were synthesized (GenScript) and subcloned in the N-terminus of dCas9 in the pCI-neo vector (Promega). The dCas9. VP64-p65-Rta (VPR) construct^{19 (link)} which served as the template for the various PCR amplifications was purchased from Addgene (#63,798; Cambridge, MA, USA). All VP-based constructs were subcloned into the pCI/neo expression vector (Promega) for uniformity. The Cas-effector with synthetic activation motif (SAM) targeting the mouse TIMP1 gene was purchased from Santa Cruz Biotechnology (sc-423402-ACT, Santa Cruz, CA, USA).

Duellman T., Doll A., Chen X., Wakamiya R, & Yang J. (2017). dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases. Metalloproteinases in medicine, 4, 63-73.

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Site-Directed Mutagenesis of HYAL1 Protein

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Wild-type HYAL1-myc-his₆ cDNA, which was subcloned into pCI-neo vector (Promega, Madison, WI), was constructed previously [19] (link). We substituted the Asn⁹⁹, Asn²¹⁶, and Asn³⁵⁰ residues in HYAL1 with Gln residues by PCR site-directed mutagenesis by using overlap extension technique [20] (link). The sequences of primers used for the mutagenesis were as follows: N99Q; 5′-TGCCCCAGCAAGCCAGCCTG-3′ (forward) and 5′-CAGGCTGGCTTGCTGGGGCA-3′ (reverse), N216Q; 5′-CTAAGCCCCCAATACACCGG-3′ (forward) and 5′-CCGGTGTATTGGGGGCTTAG-3′ (reverse), and N350Q; 5′-TTCATCCTGCAAGTGACCAG-3′ (forward) and 5′-CTGGTCACTTGCAGGATGAA-3′ (reverse).

Goto Y., Niwa Y., Suzuki T., Uematsu S., Dohmae N, & Simizu S. (2014). N-glycosylation is required for secretion and enzymatic activity of human hyaluronidase1. FEBS Open Bio, 4, 554-559.

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Plasmid Constructs and Cell Viability Assay

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To generate the pcDNA-hMIF, pcDNA-hMIF^C60S, pcDNA-hMIF^P2A, and pcDNA-hMIF^N110C vectors, the cDNA of human MIF was amplified by polymerase chain reaction (PCR) and inserted into the pcDNA3.1(-) plasmid using the BamHI and XbaI sites. pCI-hSOD1^WT, pCI-hSOD1^G93A, and pCI-hSOD1^G85R were generated by inserting human SOD1 constructs into the pCI-NEO vector (Promega), between the EcoRI and the NotI sites. hSOD1^WT–EGFP and mutant hSOD1^G93A–EGFP were obtained from Jean-Pierre Julien (Laval University, Canada).
NSC-34 cells were grown at 37 °C and 5% CO₂ in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% tetracycline-free fetal bovine serum (FBS), L-Glutamine (2 mM), and penicillin (100 U/ml)/streptomycin (0.1 mg/mL) (all reagents from Biological Industries). Transfection was performed by using TurboFect (Thermo) according to the manufacturer’s protocol. When co-transfections were performed, empty plasmids were transfected as controls. Cell viability was analyzed by using the CellTiter 96 AQ_ueous one-solution cell proliferation assay (Promega) and ELISA at 490 nm, according to the manufacturer’s protocol.

Shvil N., Banerjee V., Zoltsman G., Shani T., Kahn J., Abu-Hamad S., Papo N., Engel S., Bernhagen J, & Israelson A. (2018). MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS. Cell Death & Disease, 9(2), 107.

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Generation and Characterization of PBF Mutants

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Plasmids containing full-length PBF cDNA with or without a C-terminal haemagglutinin (HA) tag have previously been described (Stratford et al. 2005 (link)). All 10 PBF mutations were recapitulated in both HA-tagged and untagged PBF using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). FLAG-tagged PBF was generated through the ligation of an oligo encoding the FLAG sequence (DYKDDDK) into the pcDNA3.1+_PBF plasmid. The FLAG tag lies within the protein after residue E34. Previous attempts to tag PBF at the N-terminal end were unsuccessful due to the presence of a cleavable signal peptide; to overcome this, the FLAG epitope lies downstream of the cleavage site. The C51R and R140W mutations were also introduced into FLAG-tagged PBF. The NIS cDNA was housed in the pcDNA3.1+ vector with a C-terminal MYC tag (Smith et al. 2009 (link)). For stable transfections, untagged wild-type (WT) and PBF mutant cDNAs were cloned into the pCI-neo vector (Promega). Transfections were performed with TransIT-LT1 reagent (Geneflow, Lichfield, UK) following the manufacturer’s protocol at a 3:1 reagent to DNA ratio and the experiments were performed after 24–48 h.

Imruetaicharoenchoke W., Fletcher A., Lu W., Watkins R.J., Modasia B., Poole V.L., Nieto H.R., Thompson R.J., Boelaert K., Read M.L., Smith V.E, & McCabe C.J. (2017). Functional consequences of the first reported mutations of the proto-oncogene PTTG1IP/PBF. Endocrine-Related Cancer, 24(9), 459-474.

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