Notes: The authors characterized mouse neocortical interneurons that express 5-HT_3A, a ligand-gated cation channel activated by 5-hydroxytryptamine (serotonin), during embryonic development. Transgenic mice that expressed green fluorescent protein (GFP) under the control of the 5-HT_3A promoter were created. Single 5-HT_3A-expressing neurons within 300µm brain sections of transgenic mice at various stages of embryonic development were subjected to whole-cell path-clamp recordings to examine their electrophysiological properties. To confirm activation of the 5-HT_3A promoter in these cells, GFP expression was visualized by fluorescence microscopy without breaking the patch clamp seal. The contents of these single neurons then were aspirated and expelled into a 10µl reverse transcription reaction. After the reverse transcription, PCR was performed to simultaneously detect mRNAs encoding two isoforms of glutamic acid decarboxylase, three calcium-binding proteins, three neuropeptides, two transcription factors and reelin, a protein thought to be involved in neuronal migration and morphology. Two rounds of PCR using nested primers were required to detect these mRNAs. PCRs were performed using GoTaq® DNA Polymerase. Amplified products were visualized by agarose gel electrophoresis, using the 100bp DNA Ladder as a size standard. (4096)

Notes: In this paper, FuGENE® 6 or HD reagent was used to transiently transfectCHO-K1 cells. Transfection conditions were as follows: Cells were plated in 60mm dishes and transfected with FuGENE® 6 using 1.03µg of DNA. Cells were plated in 100mm dishes and transfected with FuGENE® 6 or FuGENE® HD using 6µg of DNA. 

Notes: In this paper, the authors were interested in the proteomic analysis of subcellular compartments to see if there were any protein markers for colorectal cancer (CRC) when analyzing early stage tumors and colorectal adenoma and carcinoma tissues. CRC tissue was collected and assessed for microsatellite instability and chromosomal copy number changes using the MSI Analysis System, Version 1.1, and MLPA respectively. The tumor proteins then were analyzed using MALDI-TOF/TOF MS. (4116)

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

Notes: The authors identified proteins that bind to small ubiquitin-like modification (SUMO) proteins using a yeast two-hybrid screen. The proteins ability to bind SUMO was confirmed using protein:protein interaction studies. In these studies, recombinant SUMO-1 was expressed with a His₆ tag and immobilized using MagneHis™ Ni-Particles. Putative SUMO-binding proteins were expressed with a GST tag and the proteins were co-incubated to allow them to interact. Proteins bound to immobilized SUMO-1 were eluted with Laemmli sample buffer and detected by Western blot. (4121)

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

Notes: The authors used the PowerPlex® Y System to amplify Y-STRs from 154 sexual assault cases in the Philippines. Duplicate amplifications were assembled as recommended by the manufacturer, and amplified products were detected using an ABI PRISM® 310 Genetic Analyzer. Microscopic analysis revealed sperm in 15% of the cases, and Y-STR analysis revealed male DNA in 41% of the cases. (4088)

Notes: FuGENE HD reagent was used to transiently transfect U87MG cells using a 5:2 ratio of reagent to DNA (5µl reagent, 2µg DNA). Following transfection, cells were incubated for 48 hours before use. (4412)

Notes: These authors studied the formation of centrioles in Drosophila spermatids. Genomic DNA was extracted from whole flies using the Wizard^® SV Genomic DNA Purification System. The DNA was then subjected to PCR, purified and sequenced. RNA was purified from whole flies using the SV Total RNA Isolation System. After RNA extraction, the samples were used in RT-PCR. (4064)

Notes: These authors describe a method for comprehensive expression profiling of tissue mitochondria, using swine heart as an example. After protein extraction, a 2-step trypsin digestion method was used to obtain peptides. The authors evaluated use of ProteaseMax as an alternative method to enhance digestion during the first digestion step of the process. The advantages and disadvantages of the method are discussed. (4085)

Notes: Mineral accumulation was studied in Arabidopsis thaliana comparing loci involved with growing in soil versus hydroponics. An F2 population derived from a cross between Landsberg erecta (Ler; maternal parent) and Eringsboda-1 (Eri-1; paternal parent) was propagated by single seed descent for nine successive generations in soil.
The flower buds of three plants per line were collected, and the DNA extracted using the Wizard® Magnetic 96 DNA Plant System and used for genotyping with 90 amplified fragment length polymorphism PCR (AFLP) and 39 single sequence length polymorphisms (SSLP) markers to build a genetic map of quantitative trait loci (QTL). (4136)

Notes: The authors studied the ability of JAK1 V658F and A634D mutants to activate the Janus kinase (JAK)/STAT pathway when expressed alone or together with the other components of the interleukin-9 receptor complex. The BOX1 motif of wild-type IL-9Rα, the JAK interacting region, was mutated from PXP to SXS using the GeneEditor™ in vitro Site-Directed Mutagenesis System. To assess STAT transcriptional activity, HEK293 human embryonic kidney, COS-7 monkey kidney, U4C human fibrosarcoma and g2A cells were cotransfected with 250ng of the appropriate constructs, 500ng of firefly luciferase vectors and 50ng of pRL-TK Vector and empty plasmid for a total 1.5µg of DNA. After 24 hours, the cells were lysed in 150µl of Passive Lysis Buffer and reporter activity measured using the Dual-Luciferase^® Reporter Assay System. The ProFection^® Mammalian Transfection System—Calcium Phosphate was used to transfect 10⁶ HEK293 cells in a six-well plate with 3.75µg of plasmid for Western blot analysis and cotransfected 6 × 10⁶ HEK293 cells in a 100mm dish with 14µg plasmid for immunoprecipitation studies. (4025)

Notes: The authors studied the effect of nucleoside modifications in short interfering RNA (siRNA) on 5´ phosphorylation by Clp1 kinase, binding to the Argonaute protein Ago2 and Ago2-mediated cleavage. Mice were injected with 6mg/kg of a siRNA targeting apolipoprotein B (apoB), and total RNA was isolated from the livers after 24 hours. 5´ rapid amplification of cDNA ends (5´ RACE) was used to monitor mRNA cleavage and degradation, as cleaved target RNA yields a 150bp amplification product and uncleaved RNA does not yield an amplification product under the amplification conditions. The amplification steps of the 5´ RACE protocol were performed using GoTaq® Colorless Master Mix and gene-specific primers. (4097)

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 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: These authors describe a method for cell-surface labeling with two genetically expressed tags, a biotin acceptor peptide (BAP) and HaloTag (HT). BAP and HT were fused with the N-terminus of the mouse Ig κ-chain leader sequence to direct them to the secretory pathway, and the c-terminus of the platelet-derived growth factor receptor transmembrane domain to anchor them to the plasma membrane. Fluorescent proteins were fused with BAP and HT to create the two distinct fluorescent transmembrance proteins BAP-YFP-TM and HT-CFP-TM. The constructs were introduced into HeLa cells and imaged simultaneously. The authors then demonstrated in vivo imaging by injecting the doubly-tagged HeLa cells into nude mice and imaging with near infrared fluorescent probes, and with magnetic resonance probes. (3998)

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard^® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (T_c), which is lower than the melting temperature (T_m) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq^® Flexi DNA Polymerase and mutation-specific TaqMan^® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan^_® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

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: This paper examined the role of the Cdk3/c-Jun signaling in EGF-stimulated cell proliferation and cell transformation. An AP-1 luciferase reporter plasmid construct was contransfected with various expression vector combinations of Cdk3, Cdk2, c-Jun, c-Jun M63/73, Cdk3-DN and cyclin C and the phRL-SV40 Vector as a normalization control. Cells were lysed at room temperature for 30 minutes with gentle shaking and luciferase activity measured using the Dual-Luciferase^® Reporter Assay System. SaoS-2 cells transfected with a mock and Cdk3 RNAi vector were seeded at a density of 4 x 10³ in 96-well plates with 20µl of medium. After 24 hours, 20µl of CellTiter^® 96 AQ_ueous One Solution was dispensed to each well, and the plate incubated for 1 hour at 37°C. The reaction was stopped using 25µl of 10% SDS and absorbance was measured at 492 and 690nm. For the CheckMate™ Mammalian Two-Hybrid System, the plasmid constructs, pACT-c-Jun, pBIND-Cdk3 and pG5luc, were cotransfected into HEK293 cells at no more than 100ng/well in a 48-well plate. After incubation and lysis, 20µl of lysate was dispensed into each well of a 96-well luminescence plate. Firefly and Renilla luciferase activity was detected. (4028)

Notes: The authors tested if CYP1B1 removed cellular oxygenation products that induce oxidative stress and promote the release of antiangiogenic factors. The P450-Glo™ CYP1B1 Assay was used to determine CYP1B1 activity. The presence of glutathione was assessed using either 10⁴ retinal endothelial cells or 50µl of mouse retinal extracts dispensed into each well of a 96-well plate with the GSH-Glo™ Glutathione Assay. (4010)

Notes: The protein phosphatase Cdc14 is sequestered in the nucleolus during interphase and, after successful interphase, is dispersed from the nucleolus throughout the cell by an unknown mechanism to drive a cell's exit from mitosis. The authors determined that Cdc14 contains a nuclear localization signal (NLS), but phosphorylation of serine and threonine residues adjacent to the NLS interferes with localization. These phosphorylation sites were mapped using trypsin and AspN digestion, followed by mass spectrometry. The authors showed that phosphorylation of Cdc14 is mediated by the protein kinase Dbf2-Mob1 by co-incubating purified Dbf2-Mob1 and Cdc14 in the presence of γ-[³²P]ATP. Dbf2-Mob1 was expressed with a His6 tag and purified using MagneHis™ Ni-Particles. (4120)

Notes: This paper explored the effect of methyl-CpGmodified single-stranded oligonucleotides (ssODN) on gene repair. The Wizard^® Genomic DNA Purification Kit was used to isolate gDNA from methylCpG-ssODN-treated myoblasts derived from limb muscle of neonatal C57 mice that stably expressed the mismatch repair site. One microgram of purified gDNA was digested overnight with 5U of restriction enzyme, purified, resuspended in 20µl of water and 5µl used in real-time PCR to determine if the target had been repaired. (4062)

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)