Notes: The authors used a microfabricated capillary electrophoresis instrument to rapidly assess the genetic relationship between same-sex twins and their parents and siblings. STR typing was performed to determine if the twins were monozyotic or dizygotic and to confirm familial relationships. The authors used the PowerPlex^® 16 System to examine 15 STR loci in this study. (4045)

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag^® reporter using the HaloTag^® coding region from the pHT2 promoter. (3924)

Notes: The authors of this paper describe the development of a cell-free assay system derived from Agrobacterium tumefaciens NTL4(pCF218) (pCF372), which expresses β-galactosidase under the control of a tra1 promoter. The tra1 promoter is responsive to quorum-sensing signals. The assay was developed to address time-consuming cell conditioning and incubation times of the standard whole-cell assays used to detect quorum-sensing signals in natural systems. Replacing the X-Gal substrate with the luminescent Beta-Glo^® substrate increased the assay sensitivity by tenfold. (3936)

Notes: This paper describes analysis of DNA samples at a mock crime scene using automated DNA purification followed by STR analysis on a portable microchip system. The mock crime scene was created using "victim" and "suspect" blood stains prepared by spotting 3µl of liquid blood onto paper towels and a cloth shirt. The samples were allowed to dry overnight before placement at the crime scene. Samples were processed at the scene using the Maxwell^® 16 Instrument and DNA IQ™ Casework Sample Kit for DNA extraction, and a microchip analyzer to perform amplification and STR analysis. The 9-plex autosomal STR typing system used in the microchip system included primer sequences from the PowerPlex^® 16 System (D3S1358, THO1, D21S11, D5S818, D13S317, D7S820, vWA and D8S1179) and amelogenin for sex identification. DNA purification from suspect samples was completed in 2 hours, and subsequent STR analysis and generation of a "suspect" DNA profile took a further 3 hours. The entire process from sample collection to generation of a CODIS "hit" took 6 hours. (3922)

Notes: The authors of this study used the gene module map method to identify genotype-specific therapies for human cancers. They studied breast cancer cell lines that expressed the "mitochondria" and "wound signatures". These cell lines, which are associated with tumors that have poor prognosis, were also shown to have a "proteasome signature" and were sensitive to the proteasome inhibitor bortezomib. The authors used the Proteasome-Glo™ Chymotrypsin-Like Cell-Based Assay to show that genotype-specific killing of "wound signature" cells was not due to differential degrees of proteasome inhibition, since 1 and 1µmol/L of bortezomib completely inhibited the ability of the wound signature and control cells to cleave the luminogenic substrate. More likely, the wound signature genotype conferred increased susceptibility to death from proteasome inhibition. (3870)

Notes: The authors examined the role of sxy expression in hypercompetence of Haemophilus influenza. The 1.8kb sxy gene was subcloned into the pALTER®-1 Vector and point mutations made using the Altered Sites® II in vitro Mutagenesis System. The mutated gene was sequenced and subcloned back into the original plasmid. The wildtype and mutant sxy genes were transcribed, dephosphorylated and labeled with 32P ATP. The end-labeled RNAs were then subjected to S1 nuclease mapping. The E. coli S30 Extract System for Linear Templates was used with the wildtype and mutant sxy constructs and the expression levels of the expressed protein measured by spot blotting. (3995)

Notes: In this study, A549 human lung carcinoma cells and adenovirus-transformed human embryonic kidney cells (HEK293) were transfected using FuGENE® 6 Transfection Reagent. Cells were transfected with 1.2µg of DNA at a 3:1 FuGENE:DNA ratio. (4364)

Notes: The authors determined the role of various domains of the hepatocyte growth factor activator inhibitor type 1 (HAI-1) in the inhibition of the protease matriptase. HAI-1 mutants lacking one or more domains were expressed as His-tagged fusion proteins in CHO-K1 or COS-1 cells, and proteins were purified using the HisLink™ Protein Purification Resin. Purified proteins were then used in protease assays with recombinant matriptase. (3788)

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi^® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT^® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

Notes: The authors show that SepN, a selenoprotein of unknown function, and ryanodine receptor (RyR) intracellular calcium release channel are both required for normal muscle development in zebrafish. Furthermore SepN and RyR interact, and SepN is required for full activity of the RyR channel. As part of their study, the authors expressed SepN as a polyhistidine-tagged (8X His) protein in TNT^® Coupled Reticulocyte Lysate System and purified it using the MagZ™ Protein Purification System. The TNT^® reaction was modified to optimize selenocysteine incorporation efficiency; the reaction contained 80% rabbit reticulocyte lysate (RRL), 1mM methionine, 0.4mM spermidine, 0.01µg/ml SepN-8X-His DNA and 300nM of the C-terminus of Secis Binding Protein 2 , which is required for efficient incorporation of selenocysteine in RRL-based reactions. (3898)

Notes: The authors examined the pattern of Anaplasma phagocytophilum msp2 expression, a gene that modulates with little immune pressure and has decreased virulence with prolonged in vitro passage. C57BL/6J mice were inoculated with HL-60 cells infected with low-passage (passage 5) or high-passage (passage 26). Blood samples were taken 2–21 days post-inoculation, and total RNA was isolated. The purified RNA was subjected to RT-PCR, cloned into the pGEM^®-T Easy Vector, transformed and plated. Plasmids were purified using the Wizard^® SV 96 Plasmid DNA Purification System, and the insert size analyzed after EcoRI digestion. The inserts were sequenced, aligned with A. phagocytophilum Webster strain msp2 references using ClustalX and the diversity of msp2 transcripts divided into low- or high-passage bacteria. (3975)

Notes: The authors developed seven new human embryonic stem cell (hESC) lines, five with normal karyotypes and two with abnormal karyotypes. They examined their biological characteristics, STR loci, HLA typing, differentiation capability, imprinted genes, DNA methylation and X chromosome inactivation status to determine if hESC lines with abnormal karyotypes are useful experimental tools. STR genotyping was performed using the PowerPlex® 16 System and the ABI PRISM® 3100 Genetic Analyzer. (4040)

Notes: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq^® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

Notes: This paper demonstrates use of HaloTag® technology to study expression, trafficking and translocation of an integrin-HaloTag® fusion protein. The authors fused the Halotag reporter protein to truncated integrin. They then labeled live cells with different cell-permeant and impermeant ligands and followed spatial separation of plasma membrane and internal pools of the integrin-HaloTag® protein. (3912)

Notes: The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System. (3977)

Notes: Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being subcloned into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418. (3990)

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 sequence the mitochondrial genome one of the most widespread and common collembolan species of Antarctica, springtail Cryptopygus antarcticus. Specimens were collected from Killingbeck I during a 2002 polar expedition and frozen in liquid nitrogen. The Wizard^® SV Genomic Purification System was used to extract total DNA from the samples and the complete mitochondrial genome was amplified twice, first with universal primers and sequenced, and then using long PCR with specific primers. The long PCR products were mechanically sheared, blunt end repaired and purified using the Wizard^® SV Gel and PCR Clean-Up System. The fragments were then cloned, transformed and sequenced. (3976)

Notes: The authors investigated the expression pattern of type 1a growth hormone secretagogue receptor (type 1a GHSR), a receptor for ghrelin. In situ RT-PCR was performed on paraformaldehyde-fixed, paraffin-embedded mouse spinal cord tissue. Prior to in situ RT-PCR, tissue sections were treated with proteinase K and triethanolamine, then dewaxed. Reverse transcription was performed using the Reverse Transcription System and oligo (dT)₁₅ primers; followed by amplification using the PCR Master Mix in the presence of 11-digoxygenin-dUTP (1mM). Amplification products were detected using an anti-digoxygenin, alkaline phosphatase-conjugated goat antibody and nitro blue tetrazolium/5-bromo-3-indolylphosphate-p-toluidine salt (NBT/BCIP). (3968)

Notes: Natural rubber-degrading bacteria fall into two categories: those forming clearing zones on latex overlay plates and those that do not. To investigate this degradation process, the authors amplified latex-clearing protein (lcp) homologs from non-clearing-zone-forming bacteria using degenerate PCR primers based on lcp sequences from clearing-zone forming species. The 3´ region of the lcp gene in G. westfalica was amplified by nested PCR using biotinylated primers, and the amplified products were cloned in the pGEM^®-T Easy Vector and sequenced using universal M13 forward and reverse primers. (3907)

Notes: The mutation responsible for the hairless phenotype was linked to a 80kb deletion of chromosome 7q36. Because many basic keratin genes are located at 7q36, the authors examined keratin gene expression in the Hirosaki hairless rat using RT-PCR. Expression of kb21, kb23 and kb26 was not detected, whereas other basic keratin genes were expressed. RT-PCR was performed using the AccessQuick™ RT-PCR System and 0.5µg of total RNA isolated from rat skin for 21–30 cycles. (3887)

Notes: To study the potential interactions among the five SALMs (synaptic adhesion-like molecules) family members, the cDNAs of the five SALMs were subcloned and then transiently transfected in various combinations of two to five SALMs into HEK293 cells. The cells were transfected using calcium phosphate precipitation with an equimolar amount of the pAdVAntage™ Vector to enhance protein production. Interactions of the expressed SALMs were analyzed by immunoprecipitation experiments. (3754)

Notes: These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor^® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM^®-T Easy Vector before transfer into an expression vector. (3875)

Notes: To investigate the importance of the zinc knuckle motif of early B cell factor (EBF) in DNA binding and transcription activation of target genes, the authors used a baculovirus system to express wildtype and mutated forms of EBF, then purified the proteins for use in DNA-binding assays. The EBF proteins were expressed with a biotinylated tag and purified using SoftLink™ Soft Release Avidin Resin. (3917)

Notes: These authors showed that the ETS transcription factor Erg interacts with the VE-cadherin promoter region and regulates endothelial apoptosis through this interaction. They demonstrated that inhibition of Erg by siRNA resulted in decreased VE-cadherin mRNA and protein levels, and showed that Erg interacts with the VE-cadherin promoter using a CHIP assay. To show the functional relevance of this interaction, HeLa cells were transfected with a pGL4 Vector containing the VE-cadherin promoter region and an expression vector containing Erg2 cDNA. In this reporter assay, Erg overexpression resulted in ~1.8 fold transactivation of VE-cadherin promoter activity, as measured using the Dual-Luciferase® Reporter Assay System. Inhibition of Erg in human umbilical vein endothelial cells also resulted in a loss of viability and an increase in activation of caspase 3 and caspase 7. The authors showed that apoptosis could be significantly decreased by overexpression of VE-cadherin, indicating that Erg regulates survival partially via its interaction with VE-cadherin. The Caspase-Glo® 3/7 Assay was used to measure caspase activity in these experiments. (3872)