Notes: Glucocorticoid receptor (GR) plays critical roles in cell growth, inflammation, and gluconeogenesis. Here, the regulation of GR by 14-3-3β and ϒ is characterized. Transcription of GR in the presence of 14-3-3 proteins was monitored by the β-galactosidase Enzyme Assay and the Luciferase Assay System. 14-3-3β and ϒ showed transcriptional activation of both GR, glucose-6-phosphate, and phosphoenolpyruvate carboxylase (PEPCK).  (5120)

Notes: cDNA synthesized from RNA obtained from a rat hybridoma that produced a monoclonal antibody specific to bovine PD-1 was amplified and cloned into the pGEMT®-T Easy Vector. Additionally the Wizard® Genomic DNA Purification Kit was used to purify DNA from peripheral blood mononuclear cells (PBMCs) obtained from genomic DNA was extracted from antibody-inoculated cattle. (4771)

Notes: To express the pank2 gene product, expression vectors were transfected into COS7, HeLa and SH-SY5Y cells using ViaFect™ Transfection Reagent (10⁵ cells per chamber of glass slides; 1µg expression vector DNA: 3µl ViaFect™ Reagent). The cDNA clones used in this study were amplified by RT-PCR using ImProm-II™ Reverse Transcriptase for cDNA synthesis and Pfu DNA Polymerase for PCR. PCR products were cloned into the pGEM®-T Easy Vector. (4663)

Notes: The authors demonstrate evidence of transplacental transmission of Zika virus through detection of viral proteins and viral RNA in placental tissue samples from expectant mothers infected at different stages of gestational. To confirm the identity of the flavivirus in the IHC assays, the corresponding FFPE tissue block was punched with a hollow needle, and tissue cores 3 mm in width were removed for molecular studies. Total RNA was extracted from these cores using the ReliaPrepTM FFPE total RNA Miniprep System according to the manufacturer’s recommendations. RNA was eluted in 50μL of elution buffer, and 5 μL of the extracted RNA was amplified by real-time RT-PCR using two primer/probe sets specific for ZIKV (Lanciotti et al. 2008). Real-time assays were performed using the GoTaq Probe 1-Step RTqPCR System. (4719)

Notes: PCR products from a tertiary TAIL-PCR were separated by agarose gel electrophoresis and purified using the Wizard® SV Gel and PCR Clean-Up Kit. Purified fragments were cloned into pGEM®-T Easy Vectors, and clones were sequenced. (4547)

Notes: The authors used microarray analysis, bisulfite sequencing and pyrosequencing to examine a possible link between aberrant DNA methylation and abnormal human spermatogenesis and male infertility. They identified almost 600 genes that were differentially methylated in testis tissue of men with secretory male infertility. Genomic DNA used in the microarray analysis was extracted from testicular biopsies using the Wizard® Genomic DNA Purification Kit. For the bisulfite sequencing experiments, genomic DNA was bisulfite-modified, amplified, cloned using the pGEM®-T Easy Vector System, then sequenced. (4257)

Notes: Swab samples from 11 species of Chinese bats were vortexed in maintenance medium, filtered through a 0.45µm pore filter, centrifuged and resuspended. Any nonencapsidated (naked) nucleic acid was digested in a cocktail of enzymes including 20U of RNase ONE™ Ribonuclease prior to DNA and RNA purification. Conserved regions of the RNA-dependent RNA polymerase gene of astroviruses were reverse transcribed, amplified and cloned into the pGEM®-T Easy Vector for sequencing. (4552)

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq^® Flexi DNA Polymerase, then PCR products were cloned into the pGEM^®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag^® CMV Flexi^® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag^® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag^® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase^® Reporter Assay System. (4123)

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopsies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM^®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard^® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

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: The authors investigated gene transcription within periplasmic flagella of Borrelia burgdorferi, which are composed of a basal body, hook and filament, to determine if hook formation influences flagellin gene expression. They used insertion mutagenesis to construct strains with mutated versions of the hook structural gene flgE that were disrupted by a kanamycin-resistance cassette. The flgE gene and antibiotic-resistance cassette were amplified by PCR and cloned into the pGEM^®-T Vector. To assess the effect of flgE disruption on the transcription of filament proteins FlaA and FlaB, quantitative RT-PCR was performed; enolase was used as an internal control. Negative controls without the reverse transcriptase were included for each sample. (3885)

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard^® SV Gel and PCR Clean Up System and then subcloned into the pGEM^®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

Notes: One potential source of antibiotic-resistant bacteria is food-producing animals. The authors examined the ability of protegrin-1 (PG-1), an antimicrobial peptide, to protect wildtype and transgenic mice expressing PG-1 against bacterial infection. As part of the cloning strategy to produce the PG-1 expression construct, the authors amplified and cloned full-length PG-1 into the pGEM^®-T Easy Vector. To test the bactericidal activity of PG-1 expressed in transgenic mice, radial diffusion assays were performed, in which test samples were added to a well containing E. coli and the clear antibacterial zone was measured. Two of the test samples were neutrophil secretions from the PG-1 transgenic mice and purified polyhistidine-tagged PG-1 protein, purified using the MagneHis™ Protein Purification System. (3896)

Notes: The authors characterized five open-reading frames flanking the alcohol-inducible alkane monooxygenase (BMO) structural gene of Pseudomonas butanovora. Strains with mutated bmoR, which encodes a putative transcriptional regulator, or bmoG, which encodes a putative chaperonin, were created by gene inactivation. The bmoR gene was amplified and cloned into the pGEM^®-T Vector for disruption with a kanamycin cassette. The two termini of the bmoG gene were amplified separately, ligated to the kanamycin cassette and cloned into the pGEM^®-T Easy Vector. Plasmids encoding the disrupted genes were transformed into Pseudomonas butanovora by electroporation. (3893)

Notes: To learn about the ideal target binding sequence for SATB1, the T-lineage-enriched chromatin organizer and transcription factor, random oligonucleotides underwent SELEX and five rounds of selection by EMSA. The enriched library of oligos was cloned into the pGEM®-T Easy Vector, transformed and sequenced. Several variants of SATB1-binding consensus sequences were annealed, ligated into the pGL3-Promoter Vector and cotransfected into HEK 293 cells with a plasmid that either contained SATB1 or was empty. After 48 hours, the cells were harvested and luciferase activity measured. The CheckMate™ Mammalian Two-Hybrid System was used to assess how the N-terminal PDZ domain of SATB1 interacted with the Cut and homeodomain in the C-terminus. (3982)

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: 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: 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: 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: The authors identified a novel subunit of the voltage-dependent L-type Ca²⁺ channel (Ca_V1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM^®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

Notes: The authors cloned the novel equine aldo-keto reductase AKR1C23 and characterized its expression patterns in the preovulatory follicle. The AKR1C23 cDNA was amplified from equine ovarian RNA using the Access RT-PCR System and primers designed by sequence alignments of known AKR sequences, then cloned into the pGEM^®-T Easy Vector. Levels of AKR1C23 and ribosomal protein L17a mRNAs in various equine tissues were quantified using the Access RT-PCR System and 21 cycles and 18 cycles, respectively, followed by agarose gel electrophoresis, transfer to nylon membranes, and hybridization to radiolabeled probes synthesized using the Prime-a-Gene^® Labeling System. (3791)

Notes: The authors explored the role of five different eukaryotic initiation factor (eIF) 4GI protein isoforms, which are encoded by alternatively spliced mRNAs, by using short interfering RNAs (siRNAs) to silence the eIF4GI gene. Three eIF4GI siRNA target sequences were evaluated for their ability to reduce eIF4GI mRNA levels in HeLa cells. To quantify the extent of gene silencing, a control plasmid that encodes an eIF4GI/Renilla luciferase fusion mRNA was created using the psiCHECK™-2 Vector. Cotransfection of HeLa cells with the eIF4GI siRNAs and psiCHECK™-2 control plasmid resulted in degradation of the eIF4GI/Renilla luciferase mRNA, leading to reduced Renilla luciferase activity and lower light output. The psiCHECK™-2 Vector encodes the firefly luciferase gene, which allowed normalization of Renilla luciferase expression. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase^® Reporter Assay System. Quantitative PCR (qPCR) was used to quantify the silencing of endogenous eIF4GI mRNA splice variants. Prior to qPCR, total RNA was isolated from siRNA-expressing HeLa cells, then reverse transcribed using the ImProm-II™ Reverse Transcription System. qPCR was The pGEM^®-T Easy Vector was used in the creation of plasmids encoding siRNA-resistant eIF4GI isoforms, which were transfected into siRNA-expressing HeLa cells to restore eIF4GI function. (3778)

Notes: Rat cDNA fragments of the genes encoding copper-zinc superoxide dismutase, manganese superoxide dismutase and glutathione peroxidase were amplified and cloned into the pGEM^®-T Easy Vector. The clones were used to probe Northern blots to examine the effect of age on the expression of these antioxidant defense genes. (3632)