Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify Bacillus anthracis chromosomal DNA, which was then used in PCR to amplify a luxN ortholog (named open reading frame BA5047). Amplified DNA containing the luxN ortholog region was then cloned into the pGEM^®-T Easy Vector. This construct, when transformed into Vibrio harveyi (AI-1^-, and AI-2⁺), allowed the detection of an upregulated signaling system by a bioluminescent assay. (3092)

Notes: Researchers used the pGEM®-T Vector System to clone the entire 1.4kb Shiga toxin type 2 gene (Stx2) from E. coli O157-H7 C600 (933W). The resultant construct, named pGEMTStx2, was used as a template in PCR to amplify each region of the gene corresponding to Shiga toxin type 2 subunits A and B. Each PCR product was digested with BamHI and EcoRI before ligation into pCDNA 3.1+ (Invitrogen) to create pStx2ΔA and pStx2B. Mice were then immunized with either one or both of these constructs and another construct expressing murine granulocyte-macrophage colony-stimulating factor. Expression of each subunit in mouse tissue was verified by RT-PCR with specific primers and the AccessQuick™ RT-PCR System. (2701)

Notes: Total RNA was obtained from non-small cell lung cancer cell lines and the breast cancer resistance protein message was amplified using real-time quantitative RT-PCR. RT-PCR products were subcloned into the pGEM^®-T Easy Vector. (2714)

Notes: The authors used ISSR PCR to quantitate genomic instability using matched tumor (OSCC) and normal oral squamous cell samples. The inter-repeat region bands of similar molecular size that were altered in more than one case of OSCC were reamplified, gel purified and cloned into the pGEM T Vector for sequencing. (2635)

Notes: Researchers performed a 5´ RACE analysis on the human CCR8 and mouse CX3CR1 transcripts.  PCR-amplified products from the reactions were cloned in to the pGEM^®-T Vector for further analysis.  Also, the human CCR8 and mouse CX3CR1 promoters were cloned into the pGL3-Basic Vector and transfected into THP1 and Jurkat cells. For transfections, 15 x 10⁶ cells in 750μl were used with 15μg of construct in electroporation reactions. Transfectants were grown for 48 hours and analyzed with the Bright-Glo™ Luciferase Assay System. A fourfold increase in activity was observed compared to the pGL3-Basic Vector alone. (2729)

Notes: The pGEM^®-T Easy Vector was used to subclone products of a 5´ RACE reaction. A promoter construct, assembled in the pGL3 Basic Vector, was co-transfected with a zebrafish interferon expression vector in the ZF4 zebrafish embryo fibroblast cell line using the TransFast™ Reagent (details provided). Luciferase levels were examined with the BrightGlo™ Luciferase Assay Reagent. Induction of zebrafish mRNA was also examined in zebrafish liver cells (ZFL) following treatment with the known interferon inducer, poly(I)-poly(C). RNA was extracted and reverse transcribed using ImProm-II™ Reverse Transcriptase. The resulting cDNA was used for quantitative, real-time RT-PCR with a SYBR green-based assay. (2627)

Notes: cDNA fragments from the Nogo gene were amplified from genomic DNA and cloned into the pGEM^®-T vector. The Wizard^® Plus Mini- and Midiprep kits were used to purify the plasmids from bacterial cells. (2658)

Notes: pGEM^®-T Easy Vector was used to clone PCR products.  The CytoTox 96^® Non-Radioactive Cytotoxicity Assay  was used to determine specific cytotoxicity of human dendritic cells that were transduced with recombinant adenoviruses containing the gene encoding a fusion protein of granulocyte-macrophage colony stimulating factor and carbonic anhydrase IX. (2674)

Notes: Promega T4 Polynucleotide Kinase was used to end-label an oligo representing a NF-kB binding sequence element with [γ-³²P] ATP (7,000 Ci/mmol).  Specific primers to IFN-β and IκB-α messenger RNA were used in RT-PCR to generate products for cloning into the pGEM^®-T Easy Vector. The resulting plasmids were linearized with Nco I and Spe I, respectively, and used as templates for in vitro transcription using the Riboprobe^® System to generate probes for use in an RNase protection assay.  The antisense probes were labeled with [α-³²P]CTP (800 Ci/mmol) in the Riboprobe^® reactions.  RNase ONE™ Ribonuclease was also used in this study.  (3197)

Notes: Total and poly(A)+ RNA from treated and untreated 8701-BC breast cancer tumor cells were treated with RQ1 RNase-free DNase. The RNA samples were reverse transcribed using the M-MLV RNase H– point mutant reverse transcriptase and random primers to create cDNA used in downstream analyses. The cDNA from the reactions were used in PCR, differential display PCR, and semi-quantitative multiplex PCR reactions. In differential display PCR studies, the authors analyzed differentially displayed bands by staining 6% polyacrylamide gels with the SILVER SEQUENCE™ Staining Reagents. Differentially displayed bands were re-amplified and cloned using the pGEM®-T Easy Vector and high efficiency JM109 competent cells. (3020)

Notes: A full length cDNA representing the entire genome of Diaporthe RNA virus (DaRV) was successfully cloned into the pGEM^®-T Easy Vector. The resultant construct was named pDV3. A positive strand viral RNA was then synthesized from the Sal I linearized pDV3 template. Fungi transfected viral RNA were shown to have different morphological features when compared to untransfected fungi. (2757)

Notes: The cDNA for the IL-1F7b protein was amplified from a human spleen cDNA library and directly subcloned in the pGEM^®- T Easy Vector and sequenced. The clone was transferred to a bacterial expression vector with a purification tag. The expressed protein was used to make a polyclonal rabbit antibody and antibody purification column. The cDNA for the IL-1F7b was reamplified and directly subcloned into the pTARGET™ Mammalian Expression Vector. The cDNA was stably expressed in RAW264.7 mouse monocyte/macrophage cell line.  Lysates from the cells stably expressing IL-1F7b were used to test the antibodies by western blotting. Expressing cells were also tested by immunocytochemistry. (2598)

Notes: The authors used the pGEM-T vector to clone a novel gene identified through RT-PCR analysis of human endogenous retrovirus K (HERV-K). (2469)

Notes: In this paper, the effect of the novel antibiotic GS7128 on translation was determined in cell-free systems. The effective concentration for inhibition of translation was determined for both prokaryotes and eukaryotes by translating β-galactosidase from a pGEM^® vector in E. coli S30 extracts, and luciferase control RNA in rabbit reticulocyte lysates. It was determined that GS7128 partially inhibits initiation and can fully inhibit elongation of peptides by blocking the peptidyl transferase reaction. GS7128 was shown to bind ribosomes differently than any other characterized antibiotic. GS7128 resistant mutants were not resistant to any other antimicrobial agents.  (2686)

Notes: The promoter region of human CYP19 was amplified from a lambda library and cloned into the pGEM^®-T Easy Vector for sequencing. Several mutants of this aromase cytochrome P450 promoter were created and cloned into the pGL3-Basic Vector. One microgram of the various reporter constructs along with 25ng pRL-null Vector (as an internal control) were transfected into HMEC-1 and MCF-7 cells in six-well plates. Luciferase levels were assayed 48 hours post-transfection using the Dual-Luciferase^® Reporter Assay System. To further characterize the CYP19 promoter, a 30-mer region with a GATA motif was added to 250ng HMEC-1 nuclear extract in the presence of oligonucleotide competitors or antibodies using the Gel Shift Binding 5X Buffer, then analyzed by gel electrophoresis. (3110)

Notes: In this paper, the SV Total RNA Isolation System was used to isolate total RNA from Gigaspora margarita. The authors reported isolating total RNA from 100 spores or 100mg of mycorrhizal roots. One microliter of the isolated RNA was used in RT-PCR to amplify Gigaspora margarita metallothionein (MT)-like polypeptide (GmarMT1) RNA. The researchers also cloned the GmarMT1 coding region using the pGEM^®-T Vector. (3077)

Notes: Promega reverse transcription buffer, AMV reverse transcriptase, and RNAsin^® RNAse inhibitor were used in reverse transcription reactions of total RNA from HepG2 heptatoma cells. PCR products were cloned into the pGEMT vector. (2613)

Notes: Researchers PCR amplified and cloned the Trl promoter into the pGEM^®-T vector. The Trl promoter sequence, as well as the eve promoter sequence and/or a CMV promoter, were used to make luciferase reporter constructs using the pGL3-Basic Vector. Transient transfection experiments were performed with the promoter constructs and GAGA factor expression constructs. Relative luciferase activity from each experiment was measured and compared to that of a control.  (2775)

Notes: The pGEM^®-T Easy Vector was used to clone a 12.5 kb fragment containing human kidney specific chloride channel (CLC-KB) gene fused to an enhanced green fluorescence protein (EGFP) created in a long and accurate PCR (LA-PCR) reaction.  The resultant construct was further manipulated and used to make transgenic mice.  (3131)

Notes: The authors used the pGEM-T Easy Vector to clone fragments amplified from chick Notch 1, Delta 1, Ngn 1, and Ng 2 in order to generate digoxigenin labeled antisense riboprobes. (2588)

Notes: PAI-1 is an inhibitor of tissue-type plasminogen activator (t-PA) and has been shown to have neuroprotective activity through the TGF-β pathway. The authors performed RTPCR experiments to study PAI-1 expression in cultured neurons and astrocytes. To confirm specificity of PAI-1 amplified products, the products were cloned into pGEMT vectors and sequenced. Wildtype and PAI-1 deficient astrocytes were transfected  (using the cationic lipid reagent, Transfast Transfection Reagent) with a luciferase reporter gene driven by the TGF-β1 response element (CAGA-luc) to determine if PAI-1^-/- cells could transduce TGF-β1 signal. Luciferase activity was quantitated using the Luciferase Assay Kit. (2608)

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5′ UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5′ UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM® Vector and ligated to PCR-amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc). Ten additional cDNAs were made containing a variety of changes to the 5′UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5′UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5′UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

Notes: The Access RT-PCR System was used to generate nuclear gene fragments from Chlamydomonas reinhardtii for use as probes for RNA blot hybridization assays and microarrays. RT-PCR reaction products were cloned into the pGEM^®-T Easy Vector for sequence verification and to allow easier manipulation. Details of the generation of microarray slides printed on GAPII glass slides (Corning) are provided. (2694)

Notes: Total RNA was isolated from Arabidopsis tissues using the RNAgents^® Total RNA Isolation System. The RNA was further processed into the poly(A) fraction using the PolyATtract^® mRNA Isolation System. The isolated RNA was used in Northern blot analysis. Blots were probed with a ³²P-labeled RNA probe generated from a cDNA cloned into the pGEM^®-T Easy Vector using the T7 Riboprobe^® in vitro Transcription System. Bioluminescence from transgenic plants containing a firefly luciferase reporter was visualized by spraying the plants with a solution of 5mM Beetle Luciferin, Potassium Salt in 0.1% Triton^® X-100. (2570)