Notes: The authors studied how the basal rate of protein synthesis in primary cortical neurons was affected by chronic treatment of with a variety of neurotrophic factors and cytokines. Rat eukaryotic elongation factor 2 (eEF2) was cloned by PCR then subcloned into the pCI Mammalian Expression Vector and electroporated into neurons. After 72 hours, the neurons were harvested and used in various translation assays including ribosomal transit time. (4070)

Notes: The authors created a full-length infectious cDNA clone of a genotype 3 hepatitis E virus (strain JE03-1760F) for use in cell culture. The full-length ORF2 sequence of the JE03-1760F genome was amplified and cloned into the pCI Mammalian Expression Vector. The construct was transfected into PLC/PRF/5 cells for 3 days then analyzed by Western blotting. (4029)

Notes: The authors examined the role of p53 in modulating dUTPase promoter activity. Base substitution mutations of Sp1- and E2F-binding sites in the dUTPase promoter were performed using the GeneEditor™ in vitro Site-Directed Mutagenesis System. Each mutant was confirmed by DNA sequencing. To determine growth inhibition, HCT116 human colon cancer cells were seeded in 96-well plates at 3 × 10³ cells/well and treated with 5-fluorouracil (5-FU), fluorodeoxyuridine (FUdR), oxaliplatin or in combination. After 72 hours, the CellTiter^® 96 AQ_ueous One Solution was dispensed into each well and absorbance measured. RNA was isolated from HCT116 p53+/+ and HCT116 p53-/- cells. cDNA was reverse transcribed from 200ng total RNA followed by multiplex qPCR using the Plexor™ qPCR System to amplify dUTPase, thymidylate synthase and GAPDH, a housekeeping gene. The 1.2 kb region of the dUTPase promoter upstream of the transcriptional start site was amplified by PCR and the fragment cloned into the pGL3-Basic Vector. Truncated promoters were also generated by PCR and cloned into the same vector. Drosophila SL-2 cells and HCT116 cell lines were seeded in a 24-well plate and transfected with dUTPase pGL3 promoter constructs or with pCI-Neo:p53WT, pCI-Neo:p53MUT and the empty pCI-neo Mammalian Expression Vector; all transfections included the pRL-TK Vector at a ratio of 1:10. After six hours, the cells were incubated in either fresh medium or medium containing a cytotoxic agent at the appropriate concentration. Thirty hours later, the cells were lysed, quantitated by Western blotting and 20µl of lysate analyzed with the Dual-Luciferase^® Reporter Assay System. Electrophoretic mobility shift analyses (EMSA) were performed using –64 to –91 of the dUTPase-nuclear isoform transcriptional start site in the Gel Shift Assay System. (4031)

Notes: The authors wanted to understand the role of ASAP3, an Arf GTPase-activating protein in cancer cells. FLAG- or hemagglutinin-tagged ASAP3 and GAP-deficient mutants of ASAP3 were cloned in the pCI Mammalian Expression Vector. The constructs were transfected into cells and used in immunofluorescence and Western blotting experiments. (3987)

Notes: Since tumor necrosis factor alpha (TNF-α) is known to have a protective effect on herpes simplex virus type 1 (HSV-1) infection in mouse eyes and brains, a recombinant virus constitutively expressing TNF-α was generated to see if it affected virus location and spread in the eyes and brain. Mouse TNF-α DNA was subcloned from one vector using EcoRI and placed into the pCI Mammalian Expression Vector downstream of the CMV immediate/early enhancer/promoter region. Then the CMV enhancer/promoter::TNF-α region was digested with BamHI and BglII to be placed in a third vector between the UL49 and UL50 genes of HSV-1. This construct and two controls were used to create recombinant HSV-1 particles that were then injected into the eyes of BALB/c mice. (3984)

Notes: The authors wanted to examine how the Camelpox virus (CMLV) gene 176R (v-slfn) might affect orthopoxvirus virulence since it shares sequence similarity to murine schlafen (m-slfn) proteins that inhibit T cell and fibroblast growth. HeLa cells were transiently transfected with either pCI Mammalian Expression Vector alone or pCI Mammalian Expression Vector expressing v-slfn with or without a HA tag. Cellular localization of the protein was visualized using either α-v-slfn antiserum or α-HA mAb. (3757)

Notes: The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His₆-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His₆-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric. (3786)

Notes: To understand the post-translational processing of PKDH1 protein, which has a role in human disease, a single full-length cDNA of human PKHD1 was assembled from three overlapping amplimers generated from a human kidney cDNA library. This PKHD1 cDNA was cloned into the pCI Mammalian Expression Vector, then N-terminal FLAG or EGFP tags and a C-terminal 7XMyc tag were added for detection. However, the expression level was too low, and an expression-enhancing beta-globin intronic sequence was added between the construct promoter and PKHD1 cDNA sequence. The pCI-PKHD1 construct was transfected into HEK 293 cells and the protein expression assessed using affinity tag capture and Western blotting. (3758)

Notes: To explore the role that accessory proteins may play in successfully expressing odorant receptors (ORs) on the cell surface of heterologous cells, the authors explored cotransfecting receptor-transporting proteins RTP1S, Ric8b and G_αolf with an N-terminally tagged OR to generate a functional and ligand-specific expression system. Three tags (Rho, FLAG or HA) were inserted into the NheI and EcoRI sites of the pCI Mammalian Expression Vector. Amplified OR orfs were cloned into the MluI and NotI sites of the tagged pCI Mammalian Expression Vector. The accessory proteins were subcloned into HA-pCI (RTP1S) or pCI (RTP1S, Ric8b, Hsc70t and G_αolf). For cotransfection, the N-terminal tagged OR vectors (0.8µg) and the accessory proteins (individually or in combination; 0.8µg) were transfected into HEK293T or Hana3A cells. The transfected cells were then subjected to immunocytochemistry, live-cell surface staining, permeabilized staining or FACS analysis. The Dual-Glo™ Luciferase Assay System was used to assess OR activation via CRE elements on a firefly luciferase vector. pRL-SV40 Vector was the internal control for cell viability and transfection efficiency. HEK293T or Hana3A cells were plated on 96-well plates, transfected with 1µg of CRE-Luc vector, 1µg of pRL-SV40 Vector, 5µg of OR and 1µg total for all accessory proteins (0.25µg each protein with pCI Mammalian Expression Vector to keep amount of plasmid constant). Twenty-four hours posttransfection, the medium was changed to CD293 chemically defined medium, incubated for 30 minutes at 37°C then replaced with 25µl of odorant solution in CD293 for a second incubation of 4 hours at 4°C. Then the reporter protein expression levels were measured by luminescence. (3687)

Notes: To test the feasibility of the Mycobacterium bovis Ag85B gene as a DNA vaccine, the gene was amplified by PCR, introducing EcoRI and XbaI restriction endonucleases onto the primers, digested with the restriction enzymes and ligated into the pCI Mammalian Expression Vector. After transformation, the constructs were isolated using the Wizard^® Plus Minipreps DNA Purification System and analyzed using endonuclease digestion and DNA sequencing. To confirm production of a maltose binding protein (MBP)-Ag85B fusion protein in E. coli, the cell lysate was analyzed by Western blot, stained with a rabbit anti-MBP serum and secondary antibody Anti-Rabbit IgG (Fc), AP Conjugate (1:10,000 dilution) and developed with BCIP/NBT. BALB/c mice pretreated with 10mM cardiotoxin prior to intramuscular DNA immunization using construct pCI-Ag85B, administered at days 0, 15, 30, and 45. The immunization course was 50µl of DNA at a concentration of 1µg/µl in PBS with each animal receiving a total of 100µg of plasmid DNA. Antibody response was assessed at 15 days with an ELISA assay and cytokine presence tested two weeks after immunization using splenocytes for ELISA assays and ICC. (3552)

Notes: After bioinformatics analysis of the Bacillus anthracis genome for potential vaccine targets, the 197 ORFs were selected for amplification. Using the genomic DNA of B. anthracis strain Vollum, all PCR products were generated using a 5’ primer incorporating the T7 promoter, the Kozak sequence, unique restriction enzyme sites and a start codon and a 3’ primer with a stop codon and three more unique restriction sites. The amplicons were then in vitro transcribed and translated using the TNT^® T7 Quick for PCR DNA system and radiolabeled with [³⁵S]methionine. The products were analyzed by SDS-PAGE and autoradiography. To assess the immunoreactivity of the translated products, the proteins were immunoprecipitated using anti-B. anthracis antisera. The seropositive candidate amplified ORFs were cloned into the pCI Mammalian Expression Vector using compatible RE sites. These vectors were introduced into ICR mice using a Helios gene gun system (0.5µg plasmid DNA on 1µm gold particles) and the mice immunized in 2 week intervals. (3515)

Notes: To analyze whether CD8⁺ T cell responses can be generated from gene products translated from another reading frame from the primary ORF encoded by DNA vaccines, various constructs were made using the pCI Mammalian Expression Vector. These inserts included the small hepatitis B surface antigen (HBsAg) and its 3’ untranslated sequence up to the hepatitis B virus (HBV) poly(A) sequence, which contains the sequence encoding the C-terminal Pol_344–832 fragment; the 832 residue Pol protein, and the large HBsAg in an alternative reading frame and an unrelated in frame and out of frame OVA OVA_18–385 fragment. In the case of the OVA fragment, the CMV promoter was removed and substituted with the SV40 promoter from pSI Mammalian Expression Vector. The immune response to both the primary and secondary gene products was determined using transient transfection of LMH cells followed by Western blotting or immunoprecipitation or counting CD8⁺ T cells from spleen cells. (3553)

Notes: The Wizard^® Genomic DNA Purification Kit was used to isolated genomic DNA from 5 x 10⁷ human G361 melanoma cells. The isolated DNA was used for Southern blotting. Probes were constructed with the DNA 5'-End Labeling System. The cDNA for the H11 protein was expressed in 293 HEK cells using the pCI Mammalian Expression Vector. (0075)

Notes: The pCI Mammalian Expression Vector was used for transient expression of the 150kDa TNIK protein in Phoenix-A cells, a 293 derivative. (1126)

Notes: The pCI Mammalian Expression Vector was used to express human cardiac KvLQT1 isoform 1 and KvLQT1 isoform 2 in COS-7 cells. Transfected cells were used for electrophysiology studies. (1270)

Notes: This paper describes use of the PolyATtract^® mRNA Isolation System, pCI Mammalian Expression Vector, and TNT^®  Coupled Wheat Germ Extract System for RNA isolation, gene expression and gel-shifts assays (EMSA) respectively. (1477)

Notes: The pCI Mammalian Expression Vector was used to express a dominant negative version of FADD containing only the death domain. (0678)

Notes: In this paper, various constructs of GFP-polypeptides were prepared in the pCI Mammalian Expression Vector. The constructs were expressed in HeLa cells. (1319)

Notes: Full-length 885 amino acid Germinal Center kinase-like kinase and a C-terminal deletion mutant were expressed from the pCI Mammalian Expression Vector in 293 cells. (1233)

Notes: The pCI Mammalian Expression Vector was used to express mutants of the 97kDa protein in cultured keratinocytes. The vector was also used to construct a transfection control vector, pCI-β-Gal (1030)

Notes: Several proteins were transiently expressed in the 1471.1 cell line using the pCI Mammalian Expression Vector. (0379)

Notes: Green fluorescent protein was subcloned into the pCI Mammalian Expression Vector. This construct was then used a positive control for transfection of CHO-K1 cells. (1407)

Notes: An immunoadhesin-IgG Fc fusion protein was expressed into 293 cell supernatants using the pCI Mammalian Expression Vector. The expressed protein was used for in vitro binding studies and the Fc fusion was used as a means of isolating the complex with protein A. (2060)

Notes: The pCI Mammalian Expression Vector was used to construct a hemaglutinin-tagged transient expression vector. The vector was used to express AP50 protein in 293T cells The expressed protein was reacted with a GST-fusion of CTLA-4 immobilized on glutathione beads and analyzed by Western blots with an anti-HA antibody. (0100)

Notes: The cDNAs for Kv2.1 and Kv9.3 were subcloned into the pCI Mammalian Expression Vector and microinjected into Xenopus oocytes. The proteins were expressed either separately or together and tested for interaction by immunoprecipitation. The proteins were also transiently expressed in COS-M6 cells and used for electrophysiology studies. (0552)