Notes: To construct dystrophin minigenes, genomic DNA containing a mutation in dystrophin was amplified for exons 30, 31 and 32. The three PCR fragments were combined and amplified into one product. This overlap-extension PCR generated two minigenes which were then cloned into the pGEM^®-T Vector and sequenced. After EcoR I digestion, the minigenes were ligated into the pSI Mammalian Expression Vector and transiently transfected into C2C12 cells for expression analysis. (3499)

Notes: N-terminal GST fusion proteins of portions of the type-1 S2^- Ionsitol 1,4,5-triphosphate receptor were created using the pFN2A (GST) Flexi^® Vector. The constructs were expresed in BL21 (DE3) pLysS cells and used for ATP-binding assays. (3392)

Notes: The researchers sought to determine whether PPARγ expression might direct the effects of gastrin in proliferation of colorectal cancer cells (CRC). They determined that cell line DLD-1 cells had both PPARγ and gastrin receptors. They demonstrated that gastrin stimulated CRC cell proliferation with a coincident decrease in PPARγ levels. These studies show that gastrins trophic properties could be due in part to transactivation of EGFR and phosphorylation of ERK1/2, leading to a decrease in PPARγ activation.

The authors used CellTiter 96® Aqueous One Solution Cell Proliferation Assay to measure cell growth of the CRC cell line DLD-1.

The DLD-1 cells were transiently transfected with a luciferase vector, and FuGENE® 6 Transfection Reagent was used at a DNA ratio of 3:1 in 24-well plates. To normalize for transfection efficiency, the cells were co-transfected with a β-Gal reporter construct. The Dual-Luciferase® Assay System was used to measure PPARgamma activity with values then normalized to Beta-Gal, measured with the β-Galactosidase Enzyme Assay System.  (4280)

Notes: PUF proteins act to reduce expression upon binding to the 3´UTR of target mRNA sequences. In this study, the binding of various PUF proteins (including C. elegans FBF-1) to known PUF binding sites was evaluated using a yeast three-hybrid system. For this assay, various PUF proteins fused to the Gal4 activation domain were expressed in pACT and pACT2 plasmids, and DNA oligos expressing PUF binding RNA sequences were cloned into the plasmid pIIIA/MS2-2. Assays were then performed in the yeast strain YBZ-1. β-galactosidase expression, indicating interaction between each PUF protein and the various binding sites, was measured using both solid- (colony lift) and liquid-phase assays. For the liquid-phase assays, β-galactosidase expression was measured using the Beta-Glo^® Assay System. Cells were grown in selective media to an O.D.₆₀₀ of 0.1–0.3, then mixed with an equal volume of Beta-Glo^® Assay Reagent. After one hour, luminescence was measured. Luminescence values were normalized to cell number. The results of these binding assays were used to formulate a consensus PUF binding sequence, which was then used to screen a C. elegans 3´ UTR database for potential FBF-1 target sequences. (3458)

Notes: Researchers created a recombinant infectious hematopoietic necrosis virus (IHNV_LUC) that contained the Renilla luciferase gene. The IHNV_LUC construct was tested for its ability to infect and kill rainbow trout compared to other IHNV constructs. In other studies, the EnduRen™ Live Cell Substrate was used to monitor points of infection in live fish. It was found that Renilla luciferase could be detected within four days after trout were exposed to 5 x 10⁴ PFU/ml of the IHNV_LUC construct. Areas behind the fins were demonstrated to points of viral entry and infection. (3385)

Notes: These authors showed that breast cancer metastasis suppressor 1 (BRMS1) decreases the transactivation potential of RelA/p65, which contains the transactivation domain of the antiapoptotic transcription factor NF-κB, in a non-small-cell lung cancer model. BRSM1 promotes the binding of histone deacetylase 1 (HDAC1) to RelA/p65, resulting in the deacetylation of RelA/p65 and suppression of RelA/p65 transcriptional activity. To perform in vitro deacetylation assays, the authors incubate immunoprecipitated Flag-RelA/p65 with BRSM1 and/or HDAC1 proteins, which were transcribed and translated in vitro using a TNT^® Rabbit Reticulocyte Lysate System. (3612)

Notes: Previously, the POU domain of Brn-3a was shown to interact with p53 and increase cell survival. In this article, the authors explored the possibility that Brn-3b, which shares a POU domain 95% identical to Brn-3a, may interact with p53 and affect its role in apoptosis. To test the protein:protein interaction, GST-Brn-3b fusion protein was bound to glutathione Sepharose beads and incubated with ³⁵S-methionine labeled full-length or truncated p53, prepared using the TNT^® T7 rabbit reticulocyte lysate. The luciferase control included in the kit was used as the noninteracting protein control. After washing, the bound proteins were resolved by 12% SDS-PAGE, and the bands examined by radiography. To examine the effect of Brn-3b on two p53-regulated genes, Bax and p21^cip1/waf1, ND7 cells were transiently transfected with empty vector, Brn-3b, Brn-3a or p53, or Brn-3 with p53. The reporter gene cotransfected was under the control of wildtype Bax, wildtype p21^cip1/waf1, or mutant Bax. A control vector (Renilla luciferase driven by the thymidine kinase promoter) was used for normalization. Forty-eight hours posttransfection, the cells were harvested and reporter levels assessed using the Dual-Luciferase^® Reporter Assay System. (3597)

Notes: The authors of this study investigated the effect of cellular senescence on expression of the genes that regulate circadian rhythms, specifically Per2 and BmalI. The Dual-Luciferase® Assay was used to determine activity of a PER gene reporter in senescent and young primary cultured human aortic vascular smooth muscle cells. Senescent cells activated CREB, but much more weakly than dividing cells did. (3672)

Notes: This study examined both the human and the macaque counterparts of the Mas-related genes (Mrg), a family of G-protein-coupled receptors, in assays to compare function and structure. Clones of adenylyl cyclase type II (AC2) and Gα_o were subcloned into the pSI Mammalian Expression Vector and used in the co-transfection experiments with the Mrgs. Receptor Selection and Amplification Technology (R-SAT™) was used for this comparison of the human and macaque Mrgs. Cells were plated in 96-well plates and transiently transfected with 57ng of 4 different plasmids including one of the Mrg receptors, 2ng AC2 and 30ng pSV-β-galactosidase Vector. One day post-transfection, ligands were added and after 5 days, the β-galactosidase expression was assessed. (3551)

Notes: To test if a real-time PCR assay would be able to detect Campylobacter spp. in various chicken samples, several laboratories were involved in a collaborative trial. Each lab was given 18 1ml samples, including 6 chicken neck skin samples, 6 shoe cover fecal samples and 6 cloacal swab samples. DNA was extracted from these samples using the MagneSil^® KF, Genomic System, first tested using 20µl of paramagnetic particles (PMPs) and then using 75µl PMPs. Five microliters of purified DNA was used in real-time PCR analysis. (3558)

Notes: The researchers compared the Bethesda Panel to Promega's MSI Analysis System (Cat.# MD1641) for the analysis of tissues from 34 colorectal cancers in patients with hereditary nonpolyposis colorectal cancer. (4114)

Notes: Human umbilical vein endothelial cells (HUVEC) were resuspended in CaMK extraction buffer and lysed by Dounce homogenization. CaMK activity was measured using the SignaTECT^® Calcium/Calmodulin-Dependent Protein Kinase (CaMK II) Assay. (3402)

Notes: To examine which amino acids of ArsA may be important for metalloid binding and transport, substitution mutations were introduced in the arsA gene using the Altered Sites^® II in vitro Mutagenesis System. Plasmids were purified using the Wizard^® Plus Minipreps DNA Purification System and subjected to restriction enzyme digestion. (3516)

Notes: The authors of this study investigated the basis of PARP inhibition sensitivity of cells containing mutations in BRCA1 or BRCA2 and whether the role of BRCA1 and 2 in homologous recombination might underlie the PARP inhibition sensitivity. They transfected HeLa cells with antisense constructs targeted against several proteins involved in homologous recombination and assessed sensitivity to PARP inhibition. Cells containing the RNAi constructs were treated with PARP inhibitors and blasticidin and cell viability was measured using the CellTiter-Glo^® Cell Viability Assay. Antisense inhibition of several genes including those coding for RAD51, replication protein A1 and checkpoint kinase 2 among others, resulted in sensitivity to PARP inhibition. These results indicate that the role of BRCA1 and BRCA2 in homologous recombination contributes to the sensitivity to PARP inhibition. (3595)

Notes: GoTaq^® DNA Polymerase was used in PCR to detect Lactococcus lactis phage DNA strains in whey samples. Phage DNA templates were amplified directly from DNase treated and boiled whey samples. For these reactions, the researchers use 0.25µl (1.25 units) of GoTaq^® DNA Polymerase for each 50μl reaction. Primers were designed to distinguish various strains of Lactococcus lactis phage receptor-binding protein genes. (3362)

Notes: The effector cells of a high-throughput cell-cell fusion assay were dispensed in 384-well plates and expression of the envelope gene induced. Small-molecule compounds or antibodies being tested for their ability to prevent cell fusion were added to the effector cells (carrying firefly luciferase under control of HIV-2 LTR) prior to adding the target cells. The cells were cocultured for 20–24 hours, and luciferase expression was measured using the Steady-Glo^® Luciferase Assay System. (3731)

Notes: In this study, a real-time PCR assay coupled with a DNA extraction method were used to detect staphylococcal enterotoxin (SE)-encoding genes in milk, a source of staphylococcal food poisoning. Pasteurized milk prepared with known concentrations of Staphylococcus aureus was used to generate a standard curve; experimental samples were from a staphylococcal food-poisoning outbreak that occurred in Japan in June 2000. PCR inhibition was overcome by using the following DNA purification method: a sample of milk (100µl) was added to an equal volume of 0.2M sodium hydroxide and incubated at 37°C for 20 minutes. The alkaline-treated sample was neutralized using 10µl of 3M sodium acetate (pH 5.4), extracted with 1ml of petroleum ether, and centrifuged at 13,000 × g for 10 minutes at 25°C. The aqueous phase was transferred to a fresh tube and bacterial DNA was purified from the aqueous solution using the Wizard^® SV Genomic DNA Purification System. (3677)

Notes: The CellTiter-Blue^® Cell Viability Assay was used to monitor viability of Hend murine endothelial cells and IMR90 fibroblasts in the presence of various concentrations (0.02, 0.2, 2.0 or 20 µm) of the N-terminal domain of the prokaryotic hydrogenase maturation factor (HypF-N). The cells were exposed to HypF-N for up to 24 hours before the cells were washed and cultured in fresh media. The CellTiter-Blue^® Reagent was added and the cultures incubated for 1 hour before fluorescence readings were taken. Data was expressed as percent viability compared to a control. (3476)

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: To test the significance of the C or T polymorphism at position -871 of the serum B-lymphocyte stimulator (BLyS) promoter region, the BLyS promoter was amplified, digested with Kpn I and cloned into the pGL3-Enhancer Vector. HL60 cells were then transiently transfected by electroporation using 10µg of the pGL3-BLyS-promoter constructs and 40ng of the pGL4.75[hRluc/CMV] Vector, which expresses Renilla luciferase. After 48 hours, expression of the reporter genes was assessed using the Dual-Luciferase^® Reporter Assay System. (3343)

Notes: To examine the putative binding of the poliovirus nonstructural proteins to each other, the wildtype and mutant 2B, 2C, 2BC, 3A, and 3AB regions were amplified and cloned into the CheckMate™ Mammalian Two-Hybrid System vectors, pACT and pBIND, after digestion with Sal I and Mlu I. COS cells grown in 12-well plates were transfected with a total of 1.6µg DNA. To reduce competition for transcription factors, the mix of pBIND:pACT:pG5luc plasmids was adjusted from 1:1:1 to 0.053µg pBIND, 0.053µg pACT with a higher concentration of pG5luc (1µg) and an additional 0.49µg of carrier pGEM^®-3. After 24 to 28 hours, the firefly luciferase activity was assessed using 10µl of cell lysate with 100µl Luciferase Assay Reagent and measuring the luminescence. (3492)

Notes: In this study, genomic DNA was extracted from 99 frozen thymic epithelial tumor samples using the Wizard^® SV Genomic DNA Purification System. Purified DNA was used in TaqMan SNP genotyping assays for 13 epidermal growth factor receptor (EGFR) gene mutations. (3585)

Notes: The CheckMate™ Mammalian Two-Hybrid System was used to study the association of Lyn, a tyrosine kinase, and flotillin-1, a novel constituent of lipid rafts. NIH/3T3 cells were seeded in 60mm dishes at a density of ~1 × 10⁵ cells. Each plate of cells was transfected with a total of 5.4µg of plasmid DNA (a 1:1:1 mixture of pBIND-flotillin-1 vector, pACT-Lyn vector, and pG5luc vector) by lipofection. Both mutant and wildtype proteins were used to assess the interaction. The cells were harvested, lyses and assessed for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. The results were expressed as a ratio of firefly to Renilla luciferase activity. (3491)

Notes: The BacTiter-Glo™ Microbial Cell Viability Assay was used to assess total ATP levels in Escherichia Coli strain K-12 MG165 cultures. A standard curve made from dilutions of pure rATP (Promega Cat.# P1132) was used to measure samples against E. Coli cultures treated with or without real sunlight or artificial UVA light. Cultures were added to an equal volume of BacTiter-Glo™ Reagent and luminescence was measured using a Turner BioSystems model TD-20/20 Luminometer. Data were expressed as nanograms of rATP versus fluence (UVA light dose) of cells. (3477)

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)