Notes: To examine post-transcriptional regulation of B cell lymphoma (bcl)-2 mRNA in human cell lines, the bcl-2 open reading frame was cloned into the pCI-neo Mammalian Expression Vector with a FLAG tag. This construct was transfected into U2OS human osteosarcoma cells, lysed, immunoprecipitated on Anti-FLAG-coated beads and analyzed by SDS-PAGE. A 260bp fragment containing the human c-myc 3´UTR adenine-uridine(AU)-rich element (ARE) was cloned into the pGL4.71 [hRlucP] Vector above to produce the pGL4.71PmARE plasmid. For transient expression, SK-N-BE and HEK293 cells in 96-well plates were cotransfected with 200ng of the pGL4.71 constructs (carrying c-myc or bcl-2 ARE) and 200ng of the pGL3-Control Vector. Luciferase expression was assessed using the Dual-Glo^® Luciferase Reporter Assay System. HEK293 cells were also stably cotransfected with the same pGL4.71 constructs and a vector carrying G418 resistance. Renilla luciferase expression was assessed and clones chosen with similar expression levels. (4071)

Notes: The authors explored the role of a DNA replication factor, flap endonuclease I (FEN1), in regulating telomerase activity in mammalian cells. PCR was used to add a myc tag to the N terminus of FEN1 cDNA. The amplimer was gel purified, digested with NheI and SmaI, and cloned into the same sites in the pCI-neo Mammalian Expression Vector. The insert was confirmed by sequencing. (4030)

Notes: To better understand which lysine residues in monocarboxylate transporter 1 (MCT1) were involved with binding di-isothiocyanostilbene disulfonate (DIDS), a transport inhibitor, and crosslinking to embigin, rat MCT1 and embigin were subcloned into the pCI-neo Mammalian Expression Vector via the EcoRI site. The vector was then subjected to site-directed mutatgenesis of each of six lysines either singly or in combination, and the effect on the binding activity or inhibition was tested. (4069)

Notes: In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting. (3986)

Notes: The C-terminus of free methionine-R-sulfoxide reductase (fRMsr) gene, a yeast gene that can generate methionine from methionine-S-sulfoxide and methionine-R-sulfoxide, was tagged with six histidines before being cloned into the pCI-neo Mammalian Expression Vector. This construct was transfected into SK-Hep1 hepatocytes with or without the Clonegene pEGFP-N1 Vector. Using 800 μg/ml G418 sulfate, a stable cell line was selected. This cell line was then tested for response to oxidative stress. (3985)

Notes: To test subunit-dependent α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, the GluR2 KO mouse (GluR2^–/–) was used to test responses when GluR2 was introduced. Enhanced GFP was added to the N-terminus of the GluR2 subunit and GluR2 subunit constructs which were then cloned into the pCI-neo Mammalian Expression Vector. Hippocampal slice cultures were taken from GluR2 KO mice and biolistically transfected with the GFP-GluR2 constructs. Whole-cell electrophysiological recordings were taken 3–7 days posttransfection. (3759)

Notes: To examine the effects of hormones on Gas6, a plasma vitamin K-dependent protein that may play a role in cardiovascular disease, the authors developed an ELISA test for Gas6, which they tested on blood from male and female volunteers. A recombinant Gas6 control was developed by reverse transcribing the full-length human Gas6 mRNA from human umbilical vein endothelial cells, amplifying the cDNA using nested PCR and after restriction digestion, ligating the insert into the EcoRI and XbaI sites of the pCI-neo Mammalian Expression Vector. The full-length construct was confirmed by sequencing and then tested in the Gas6 ELISA. (3688)

Notes: Exploring the possibility of generating a vaccine against Neisseria meningitidis serogroup B (MenB), the authors of this study tested several antigenic sequences in mice for an anti-MenB response. The DNA vaccine was developed using the pCI-neo Mammalian Expression Vector with inserts composed of synthesized double-stranded oligonucleotides that coded for the desired peptides selected using phage display libraries. A leader sequence or a tetanus toxin universal T cell helper epitope sequence or both were inserted at 5’ of the vector multiple cloning region to obtain secretory or T cell helper peptides. Since IFN-γ was helpful as an adjuvant, a second vector was generated using pCI-neo Mammalian Expression Vector encoding murine IFN-γ was used for coimmunization in the DNA vaccine experiments. The plasmids were grown and isolated using a low-endotoxin plasmid purification system and functional peptide was analyzed by FACS. BALB/c mice (5–7 wk old) were immunized with 150µg of purified DNA (70µg of the vectors expressing IFN-γ and 70µg of the immunizing plasmid). An ELISA was used to determine various antibody levels from mouse blood. (3689)

Notes: To express cutinase on the cell surface of mammalian cells, a multidomain chimeric construct consisting of Fusarium solani pisi cutinase–hemagglutinin (HA) nonapeptide epitope tag–human fractalkine stalk–β1 integrin was cloned into the pCI-neo Mammalian Expression Vector using the enzymes NheI and NotI. CHO cells were transfected with the construct for 24 hours and sorted by FACS 10 days post-transfection using a fluorescent anti-HA antibody. After an additional 15 days of propagation, the cells were sorted a second time and determined to exhibited >85% stable transfection, as measured by immunostaining and fluorescence microscopy. (3512)

Notes: A proline oxidase (POX) antisense vector was generated by amplifying part of the POX cDNA and ligating the product into the pCI Mammalian Expression Vector in the antisense orientation. This construct was tested and validated for blocking POX mRNA expression using RT-PCR. Both PPARγ and p53 cDNAs were also cloned into the pCI Vector. The human POX promoter sequence was amplified and cloned into the NheI and HindIII sites of the pGL3-Basic Vector to create the POX-Luc reporter construct. Using several colon cancer cell lines (HT29, LoVo, HCT116, HCT15, RKO, KM12, HCC2998 and SW620), the POX-Luc construct was co-transfected with pRL-null (to normalize transfection efficiency) plus PPARγ, p53 constructs or empty vector. A PPARγ ligand was added 10 hours post-transfection and cells harvested 24–36 hours after transfection. POX promoter luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System and a TD-20/20 luminometer. (3514)

Notes: To examine the putative role that SUN1, a nuclear envelope (NE) protein with a C-terminal SUN (Sad1/UNC-84 homology) domain, may play in the inner nuclear membrane, the murine SUN1 cDNA was subcloned into the pCI-neo Mammalian Expression Vector. Primers containing the myc or HA tag sequence were used to amplify the pCI-neo construct, adding tags both 5’ and 3’ of the SUN1 cDNA. Emerin, a marker for the NE lumen, was also subcloned into the pCI-neo Vector and a 3’ myc tag added. These constructs were translated and labeled with ³⁵S in vitro using the TNT^® T7 Quick Coupled Transcription/Translation System, and the radiolabeled proteins were used in a GST pull-down assay. In addition, NIH/3T3 cells were transiently transfected with myc-SUN1 constructs, fixed in methanol and then co-stained with anti-myc and anti-lamin A/C antibodies. (3511)

Notes: Constructs of Rabex-5, a guanine nucleotide exchange factor, and Rabaptin-5, a multivalent adaptor protein, were created for transfection. The plasmid pGEM-Myc-human Rabaptin-5 was digested using NcoI/PstI, the fragment filled in using T4 DNA polymerase and ligated into SmaI-cut pCI-neo Mammalian Expression Vector. This new construct was digested with ApaI and NotI to remove Rabaptin-5 while Rabex-5 was digested using SacII/SpeI. Both digestion products were subjected to T4 DNA polymerase and the blunt-ended fragments ligated to create pCI-neo-Myc-Rabex-5. Furthermore, the Rabex-5 construct was subjected to site-directed mutatgenesis. HeLa cells were transiently transfected using the various pCI-neo-Myc-Rabex-5 vectors and the cells extracts immunoprecipitated to determine if ubiquitination of Myc-Rabex-5 had occurred. (3513)

Notes: These authors investigated the relationship between the CD3zeta protein and the TCRαβCD3εδγ complex after T-cell activation. Several of the mutant CD3zeta coding sequences used in these studies were cloned into the pCI-neo Mammalian Expression Vector, and T-cell hybridomas were transfected with these constructs to express mutant proteins. The amounts of CD3 associated with the TCRαβCD3εδγ complex at the cell surface was quantitated by biotinylating cell-surface proteins, purifying the biotinylated proteins with the SoftLink™ Soft Release Avidin Resin, immunoprecipitating the complex with an anti-TCR-Cβ antibody, then detecting the protein by Western blot analysis using a streptavidin-alkaline phosphatase conjugate. (3662)

Notes: The authors seek to determine the role of glutamate in excitotoxicity and neurofilament accumulation seen in some neurodegenerative diseases. Neurofilament light, middle, and heavy chains were expressed from rat cDNAs cloned into the pCI-neo Mammalian Expression vector in SW-13 cells. Primary rat cortical neurons were transfected with a neurofilament middle chain and green fluorescent fusion protein. SW13 cells and primary rat cortical neurons were transfected with the ProFection® Mammalian Transfection System–Calcium Phosphate. The CytoTox 96® Non-Radioactive Cytotoxicity Assay was used to monitor glutamate toxicity in these cell. To determine the role of the MAPK and JNK signaling pathways,  SW13- cells and primary neuronal cells were immunostained for dually phosphorylated MAPK and JNK using Promega's Anti-ACTIVE® MAPK pAb and Anti-ACTIVE® JNK pAb, respectively. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton® X-100 in PBS, blocked with  0.2% Tween® 20 in TBS, and incubated with primary antibodies diluted in blocking solution. Western blot analyses were performed on the primary cortical neurons to quantitate the level of dually phosphorylated MAPK protein  The blots were also probed with a pan MAPK antibody that detects total (active and inactive) MAPK. (2382)

Notes: Wildtype and mutated DHCR7 cDNAs fused with an N-terminal c-myc epitope were subcloned into the  pCI-neo Mammalian Expression Vector and were heterologously expressed in the HEK293-derived cell line tsA-201. (0144)

Notes: The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce as many as three point mutations into the same cDNA. Two deletion mutants were also introduced into the tyrosinase cDNA by the insertion of premature stop codons. The mutations were either performed in the pCI Mammalian Expression Vector or the vector pCDM8.1. (1382)

Notes: The full-length, 477 amino acid glucagon receptor was expressed in COS cells using the pCI-neo Mammalian Expression Vector. Membranes from these cells were used for binding studies. (1351)

Notes: The pCI-neo Mammalian Expression Vector was used to express the 750 amino acid Mint2 protein as well as the 695 amino acid amyloid precursor protein. Both were expressed together in CHO cells. (0715)

Notes: The RNAgents^® Total RNA Isolation System was used to isolate total RNA from a mouse hybridoma. The isolated RNA was used for RT-PCR of the variable regions of both heavy and light chains. The pCI-neo Mammalian Expression Vector was used to express the μ heavy chain in H chain-deficient hybridomas. (1412)

Notes: The pCI-neo Mammalian Expression Vector was used to make a bicistronic expression vector to express a 97kDa portion of TRP3 and an IRES sequence to express GFP. The resulting plasmid was used for expression in bovine pulmonary artery endothelial-derived cells, CPAE. Addendum to plasmid construct published in J. Physiol. (Lond) 519.3, 923. (0954)

Notes: A PCR product amplified from a mutant RB (retinoblastoma protein) cDNA was cloned into the pCI-neo Mammalian Expression Vector. In vivo cyclin-mediated phosphorylation was assayed by cotransfecting the RB expression plasmids with members of the cyclin D or cyclin E family into an RB (-/-) cell line. (0571)

Notes: Genomic DNA was purified from blood or cultured fibroblasts, by means of the Wizard^® Genomic DNA Purification Kit. Total RNA was isolated from cultured fibroblasts by use of the SV Total RNA Isolation System, and it was amplified by Access RT-PCR System by use of exonic primers. An expression vector containing an S-tag attached to the 5´ end of the FALDH cDNA was constructed using pCI-neo Mammalian Expression Vector. (0482)

Notes: Various sequences of the synaptic scaffolding molecule (SCAM) were cloned into the pCI-neo Mammalian Expression Vector. Also, a myc tag was engineered into the vector along with the SCAM sequences. These sequences were expressed in vitro with the TNT^® T7 Quick Coupled Reticulocyte Lysate System. Extracts were prepared from COS-1 cells expressing either neurabin-1, SAPAP-1, SAPAP-2, SAPAP-3 or SAPAP-4. The extracts were separated by SDS-PAGE and transferred to nitrocellulose. The [³⁵S]met-labeled, myc-tagged proteins were reacted with the COS-1 cell extracts and interaction detected by autoradiography as well as immunoblotting with an anti-myc antibody. The method of this overlay assay was referenced. (1046)

Notes: The paper uses the SV40 late polyadenylation signal from the pCI neo Mammalian Expression Vector to make transgene expressing IL-2R α/GFP fusion. (0169)

Notes: The pCI-neo Mammalian Expression Vector was used to express human c-myc in Cos cells. The plasmid was also used as a template in TNT^® Coupled Wheat Germ System for in vitro studies. Both myc proteins and E6 proteins were expressed in vitro using the TNT^® Coupled Reticulocyte Lysate System for in vitro studies. Proteins expressed in the in vitro system were partially purified over a DEAE column prior to being assayed. (1077)