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)

Notes: The authors analyzed the transcriptional activity of wild-type Chlamydomonas reinhardtii cultures sampled at different time points during the aerobic and anaerobic phase of the photobiological hydrogen production process under sulfur-depleted conditions. C. reinhardtii were grown in photobioreactors, carefully extracted, centrifuged and flash-frozen in liquid nitrogen. RNA was purified using the SV Total RNA Isolation System following the plant centrifugation protocol without sample grinding. The eluted RNA was quantitated and integrity checked by gel electrophoresis and qRT-PCR. Total RNA was used to synthesize labeled cDNA using the ChipShot™ Indirect Labeling and Clean-Up System. The labeled cDNA was used for probing microarrays. (4022)

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 infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples. (3888)

Notes: The authors identified a Y-chromosome-specific polymorphism, the M293 mutation, that defines the Y haplotype Eb1f-M293. They examined the geographic distribution of this and other Y haplotypes to determine the date of origin of the E3b1f-M293 haplotype and track the migration of the pastoralist lifestyle in Africa. Y-STR analysis was performed using the PowerPlex® Y System and two additional sets of primers. (4039)

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

Notes: PDILT is a protein disulfide isomerase (PDI) homolog under developmental control and is induced during puberty. To determine whether PDILT expression coincides with the first wave of spermatogenesis, the authors examined expression of PDILT in 2-, 15-, 30-, 45- and 58-day old rats using RT-PCR. Total RNA was isolated from rat testis, and 50ng was amplified using the AccessQuick™ RT-PCR System and primer pairs specific for PDILT, PDI and calmegin, a testis-specific chaperone that is expressed upon the appearance of spermatocytes. Primer pairs were designed to span introns to avoid amplification of genomic DNA. PCR products were analyzed on a 1% agarose gel. Results showed that PDILT mRNA was first detected during the onset of spermatogenesis. (3765)

Notes: The authors studied the role of the Erk1/2 signaling pathway during neural specification in mouse embryonic stem (ES) cells. Undifferentiated ES cells express high levels of the pluripotent marker Nanog but do not express fibroblast growth factor (Fgf5), an early marker of differentiation of ES cells, or Sox1, an early neural transcription factor gene. Using quantitative PCR, levels of Nanog, Fgf5 and Sox1 mRNA were quantitated during ES differentiation in the presence and absence of a MEK inhibitor. Prior to quantitative PCR, 1µg of total RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase. By measuring these mRNA levels, the authors determined that inhibition of the Erk1/2 pathway blocked ES differentiation. (3726)

Notes: These authors screened a library of 15,000 expression cDNAs in neuroblastoma IMR-32 cells searching for genes that activate the antioxidant response element, ARE. ARE is a cis-acting enhancer element found in the 5´ flanking region of many genes that are involved in protection from oxidative stress. The library was screened using a luciferase reporter construct under the control of an ARE-containing promoter. Luminescence, indicating the presence of cDNA-activating ARE, was measured using the Bright-Glo™ Luciferase Assay System. cDNA clones showing reproducible activation were selected for further analysis. The authors tested the effect of over expression of these ARE activators on the ability to resist oxidative stress. IMR-32 cells expressing the various cDNAs were exposed to hydrogen peroxide or rotenone, and the effect on cell viability was measured using the CellTiter-Glo^® Assay. Cells overexpressing the ARE-activators were more resistant to oxidative stress than controls. (3629)

Notes: These authors used the Kinase-Glo^® Luminescent Kinase Assay to perform a high-throughput screening assay for inhibitors of glycogen synthase kinase-3β in 96-well plates. They used a 1µM ATP concentration and screened 55,000 compounds at 10µM. The assay sensitivity and IC₅₀ values of reference compounds were comparable to radioactive methods; the final optimized assay had an average Z´-factor of 0.72. (3591)