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Sci. Rep. 16, 4694. Imaging of conditional gene silencing in vivo using a bioluminescence-based method with thermo-inducible microRNAs 2018

Pinel, K., Genevois, C., Debeissat, C., Couillaud, F.

Notes: A novel therapeutic method utilizing synthetic microRNAs combined with a heat shock-inducible promoter to decrease target gene expression is described. This method is ideal for diseases where target gene overexpression leads to disease state. To monitor gene expression, the firefly luciferase gene within a tumor is targeted and expression is monitored using the Dual-Luciferase® Reporter Assay System. The ViviRen™ Live Cell Substrate is used to monitor expression in live mice. (5088)

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Gene 641, 151–60. Individual effects of the copia and gypsy enhancer and insulator on chromatin marks, eRNA synthesis, and binding of insulator proteins in transfected genetic constructs 2018

Fedoseeva, D.M., Kretova, O.V., Gorbacheva, M.A., Tchurikov, N.A.

Notes: The function of enhancer and insulator elements for the mobile elements copia and gypsy are characterized. To determine the function of these elements, cells were transfected with luciferase constructs with enhancer and insulator elements using the TransFast™ Transfection Reagent. Luciferase activity was monitored with the Dual-Luciferase® Reporter Assay. Enhancers stimulated the synthesis of long enhancer RNAs (eRNAs) and histone methylation and acetylation marks. The presence of insulator sequence reduced eRNA synthesis. (5089)

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J. Biol. Chem. 293, 6517–29. The cellular stress proteins CHCHD10 and MNRR1 (CHCHD2): Partners in mitochondrial and nuclear function and dysfunction 2018

Purandare, N., Somayajulu, M., Hüttemann, M., Grossman, L.I., Aras, S.

Notes: Coiled-coil-helix-coiled-coil-helix domain-containing 10 (CHCHD10) protein mutations are associated with multiple neurodegenerative diseases. Here, the cellular role of CHCHD10 in oxidative phosphorylation in the nucleus and mitochondria is investigated. CHCHD10 is shown to be a regulator of genes containing the oxygen response element (ORE) using the Dual-Luciferase® Reporter Assay. Interestingly, two disease variants are shown to lead to reduced respiration and increased reactive oxygen species (ROS). (5090)

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J. Biol. Chem. 282, 7982–7890. C/EBPbeta participates in regulating transcription of the p53 gene in response to mitogen stimulation. 2007

Boggs, K. and Reisman, D.

Notes: To explore the role of C/EBPb isoforms in regulating p53 expression during the cell cycle, the 1.7 kb murine p53 promoter was cloned into the pGL3-Basic Vector. Using TransFast™ Reagent, Swiss3T3 and 6629 (C/EBPb-null) cells were transfected with 0.1–0.75 µg of pGL3-1.7-kb p53 promoter construct with or without co-transfection of 0.25 µg of C/EBPb-2, and with 50 ng of pRL-TK Vector as an internal control. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity, normalizing reporter activity to Renilla luciferase activity. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to either mutate or delete the –972/–953 cis-acting element carrying the C/EBPb-binding site within the p53 promoter, and 0.1–0.75 µg of the mutant constructs were then tested in the same reporter assay. The C/EBPb-1, -2, and -3 cDNAs were cloned into an expression vector and translated using 0.5µg of plasmid in the TNT® T7 Quick Coupled transcription/translation system either in the presence of unlabeled or [35S]methionine. The synthesized proteins were analyzed on 12% SDS-polyacrylamide gel. (3675)

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Protein Sci. 16, 82–91. Human lysosomal DNase IIα contains two requisite PLD-signature (HxK) motifs: evidence for a pseudodimeric structure of the active enzyme species. 2007

Schäfer, P., Cymerman, I.A., Bujnicki, J.M. and Meiss, G.

Notes: The authors used biochemical and mutational analysis to characterize human lysosomal DNaseIIα. Native DNaseIIα and site-directed mutants were expressed as Flag-His6-DNaseIIα and HA-tagged DNaseIIα using expression constructs created with the pCI Vector. Protein was expressed by transiently transfecting HEK 293-T cells using the TransFast™ Transfection Reagent. Flag-His6-DNaseIIα was purified using the MagneHis™ Ni-Particles, and this purified protein was used in nuclease assays to monitor catalytic activity and in gel filtration experiments and coimmunoprecipitation assays with HA-DNaseIIα to determine whether the active enzyme is monomeric. (3786)

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Proc. Natl. Acad. Sci. USA 103, 4552-4557. A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample. 2006

Guindalini, C., Howard, M., Haddley, K., Laranjeira, R., Collier, D., Ammar, N., Craig, I., O'Gara, C., Bubb, V.J., Greenwood, T., Kelsoe, J., Asherson, P., Murray, R.M., Castelo, A., Quinn, J.P., Vallada, H., and Breen, G.

Notes: These authors investigated the effect of various polymorphisms in the dopamine transporter gene (SLC6A3) on susceptibility to cocaine addiction. Genotyping of various polymorphisms in cocaine abusers and control subjects revealed a potential association of the int8 VNTR with cocaine abuse. Seven alleles of the int8 VNTR were sequenced. Various allelic sequences were then cloned into a modified phRL-SV40 Renilla luciferase reporter vector and transfected into the mouse SN4741 cell line, which expresses the dopamine transporter, and the effects on reporter activity were monitored. Sequences of two alleles were then cloned into a pGL3 Promoter Vector construct and transfected into JAP cells. The cells were then challenged with various amounts of cocaine, KCL or KCl and forskolin, and the effect on reporter activity was monitored. The TransFast™ Reagent was used for transfections at a 2:1 reagent:DNA ratio. (3543)

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J. Biol. Chem. 281, 7364-73. Human arsenic methyltransferase (AS3MT) pharmacogenetics. Gene resequencing and functional genomics studies. 2006

Wood, T.C., Salavagionne, O.E., Mukherjee, B., Wang, L., Klumpp, A.F., Thomae, B.A., Eckloff, B.W., Schaid, D.J., Wieben, E.D. and Weinshilboum, R.M.

Notes: Human arsenic methyltransferase (AS3MT) was cloned and mutated to produce allozymes for further analysis. COS-1 cells were transfected with constructs encoding the wildtype enzyme and the synthetic allozymes using TransFast™ Transfection Reagent. The wildtype enzyme and allozymes were transcribed and translated using TNT® T7 Coupled Reticulocyte Lysate System in the presence of RNasin® Ribonuclease Inhibitor. (3383)

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J. Virol. 80, 6669-6677. Human papillomavirus e7 oncoprotein dysregulates steroid receptor coactivator 1 localization and function.

Baldwin, A., Huh, K-W. and Mϋnger, K.

Notes: The MagneGST™ Protein Purification System was used to purify GST fusion proteins of the oncoprotein HPV16 E7 or various mutants of HPV16 E7. The purified GST fusion proteins were used for in vitro binding experiments with steroid receptor coactivator 1 (SRC-1), which was produced using the TNT® T7 Coupled Wheat Germ Extract System and labeled with the Transcend™ Non-Radioactive Translation Detection System. GST pull-down assays were resolved by Western analysis using streptavidn-horseradish peroxidase and alpha-GST. To determine the effects of endogenously expressed HPV16 E7 on SCR-1-mediated transcription, luciferase reporters under the control of either the IL-8 promoter or an artificial promoter containing three estrogen response elements repeats (3 × EREs) were cotransfected with a Renilla control vector into two human cervical cancer lines (C33A and CaSki) using either the Transfast™ Transfection Reagent or another commercial transfection reagent. The Dual-Luciferase® Reporter Assay was then used to determine luciferase activity to functionally map the E7-interacting domain and to determine the effects of high- and low-risk PHV E7s on SRC-1-mediated transcription. (3459)

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J. Biol. Chem. 281, 13915–13921. NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling. 2006

Suzuki, A., Ogura, T. and Esumi, H.

Notes: A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 104/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector. (3438)

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Nucl. Acids Res. 33, 3245-3252. Visualizing the dynamic behavior of poliovirus plus-strand RNA in living host cells. 2005

Cui, Z-Q., Zhang, Z-P., Zhang, X-E., Wen, J-K., Zhou, Y-F., and Xie, W-H.

Notes: Molecular beacon oligonucleotides are stem-loop oligonucleotide probes labeled with a fluorophore at one end and a quencher at the other. Upon binding to a specific target DNA or RNA, the fluorophore and quencher are separated, and the target DNA or RNA can be detected by fluorescence. In this study, the location of poliovirus plus-strand RNA was monitored in infected Vero cells using such oligonucleotide probes. The TransFast™ Reagent was used to deliver the oligonucleotide probes to the target cells. A transfection mixture containing 9µg of TransFast™ Reagent and 2µg of probe in 0.8ml HEPES buffer was incubated at room temperature for 10–15 minutes before addition to cells from which the growth medium had been removed. The transfection mixture was removed after incubation at 35°C for 40–50 minutes and replaced with fresh growth media. (3544)

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J. Cell Biol. 166, 61–71. Mutations in sticky lead to defective organization of the contractile ring during cytokinesis and are enhanced by Rho and suppressed by Rac. 2004

D’Avino, P.P., Savoian, M.S., and Glover, D.M.

Notes: The entire sti gene (~7kb) of Drosophila was PCR cloned into the pGEM®-T Vector. The cloned sequences were then sequenced and analyzed for mutations. The researchers also used the T7 RiboMAX™ Large Scale RNA Production System to create dsRNAs from PCR-amplified regions of the sti and GFP coding regions. TransFast™ Transfection Reagent was used to transfect 2 x 106 Schneider S2 cells with 10 μg of dsRNA in a 35mm Petri dish. The cells were fixed after transfection and examined for multinucleate cells, or immunocytochemically stained for actin and anillin. (3259)

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Biochem. Biophys. Res. Commun. 312, 531–536. Gonadotropin-releasing hormone gene products downregulate the expression of their neighboring genes that encode protein tyrosine phosphatases α and ε. 2003

Okubo, K. and Aida, K.

Notes: COS-7 cells were transiently transfected with vectors that express Protein Tyrosine Phosphatase α (PTPα) and Protein Tyrosine Phosphatase ε (PTPε) using the TransFast™ Transfection Reagent. Transfections were performed in 100mm dishes. The authors also used the Tyrosine Phosphatase Assay System to assay Tyrosine Phosphatase activity in the transfectants. For these assays, both Tyrosine Phosphopeptides were demonstrated to be substrates for PTPα and PTPε.  Data were expressed as percent of untransfected control.  (2856)

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J. Virol. 77, 1992-2002. Molecular and functional analysis of an interferon gene from the zebrafish, Danio rerio. 2003

Altmann, S.M., Mellon, M.T., Distel, D.L. and Kim, C.H.

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)

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Nucl. Acids Res. 31, e27 (1-7). Targeted gene modification in mismatch-repair-deficient embryonic stem cells by single-stranded DNA oligonucleotides. 2003

Dekker, M., Brouwers, C. and te Riele, H.

Notes: Mismatch-repair deficient murine embryonic stem (ES) cells that had previously undergone recombination to have a dysfunctional neomycin resistance gene inserted into their genome were transfected with either a neomycin repair oligo or a control oligo.  Transfections were performed with either Tfx™-50 or TransFast™ Transfection Reagent.  Cells were seeded in six-well plates at a density of 7 x 105 cells per well. The following day, cells were transfected for 1 hour with 1.4 ml of serum-free medium containing 3-7μg of oligonucleotide plus 31.5 μl Tfx™-50 or 63 μl TransFast™ Transfection Reagent.  The results are presented as percent efficiency of repair of the neomycin resistance gene as dictated by the presence of G418 resistant colonies. (3188)

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Nucl. Acids Res. 30, 3045-51. The effect of ICAD-S on the formation and intracellular distribution of nucleolytically active capase-activated DNase. 2002

Scholz, S.R., Korn, C., Gimadutdinow, O., Knoblauch, M., Pingoud, A. and Meiss, G.

Notes: Co-expression of murine CAD with either ICAD-L or -S in E. coli leads to a functional DFF complex, which after caspase-3 activation, releases a functionally active DNase. (2514)

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J. Biol. Chem. 277, 30798-30804. Transcription factor AP-2 interacts with the SUMO-conjugating enzyme UBC9 and is sumolated in vivo. 2002

Eloranta, J. J., and Hurst, H.C.

Notes: The authors examined how the transcription factor AP-2 interacts with and is affected by UBC9, a SUMO-conjugating enzyme. Promega's GeneEditor™ in vitro Site-Directed Mutagenesis System was used to create a single amino acid change (K10R) in AP-2γ once it was determined that the lysine was required for sumolation in vivo. Transient transfection assays were performed using the TransFast™ Transfection Reagent. All cell lines (MDA MB 436, MCF7, T47D and HepG2) were transfected in 12-well plates when 60-70% confluent. Various amounts of plasmid were used during transfection and detailed in the paper. For reporter genes, the AP-2 firefly reporter (3xAP2-Bluc) and Promega's pRL-TK Vector were used in a 1:1 ratio, the Renilla luciferase used to normalize firefly expression.  Lysates were made 48 hours post-transfection and assayed using the Dual-Luciferase® Reporter Assay System. (2558)

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Gene Ther. 7, 2028-2035. Human dendritic cells transfected with either RNA or DNA encoding influenza matrix protein M1 differ in their ability to stimulate cytotoxic T lymphocytes. 2000

Strobel, I., Berchtold, S., Gotze, A., Schulze, U., Schuler, G. and Steinkasserer, A.

Notes: In this paper, the Transfast™ and Tfx™-50 Transfection Reagents were compared to a variety of chemical transfection reagents and an electroporation procedure in their ability to transfect dendritic cells with a green fluorescent protein (GFP)- expressing vector or mRNA. Microscopic and FACS analyses were performed to determine transfection efficacy (percent of GFP expressing cells). Best results were obtained using the Transfast™ Transfection Reagent, which had a mean efficacy of 16.2% and 4.6% when transfecting mRNA and the GFP vector, respectively. Data demonstrating that dendritic cell transfection with Transfast™ Reagent helped in the process of cell maturation and antigen presentation are also presented. For all transfections with Transfast™ Reagent, 400μl of Opti-MEM medium was pre-incubated with 12μl of the transfection reagent and 4μg of vector DNA or RNA at room temperature for 15 minutes. One million cells were then added to these mixtures and the transfections were incubated at 37°C for 1 hour. The cells were resuspended, seeded in six well plates, and supplemented with appropriate cytokines. (2684)

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Neuropharmacology 38, 793-803. A simple method to transfer plasmid DNA into neuronal primary cultures: Functional expression of the mGlu5 receptor in cerebellar granule cells. 1999

Ango, F., Albani-Torregrossa, S., Joly, C., Robbe, D., Michel, J.-M., Pin, J.-P., Bochaert, J. and Fagni, L.

Notes: The authors investigated transfection of primary mammalian neuronal cultures with seven commercially available cationic lipid reagents. Green fluorescent protein (GFP) was used as the reporter and successful transfection was judged by the percentage of cells transfected and the toxicity of the transfection. Mouse cerebellar neuron cultures were grown on poly-L-ornithine-coated glass cover slips (~1 x 105 cells total; 95% granule cells and 5% non-granule cells) for initial optimization experiments. Transfections were optimized for the transfection reagent:DNA ratio and increasing amounts of DNA at the optimized ratio. TransFast™ Transfection Reagent provided the greatest transfection efficiency with an overall transfection efficiency of 5% (2% granule cells/3% non-granule cells) with no toxicity. The Tfx™-50 Reagent ranked second and was also non-toxic. TransFast™ Reagent was most effective at a 1:1 ratio (3µl TransFast™ Reagent/µg DNA) prepared in 1ml of serum-free medium. The TransFast™ Reagent method was also tested on cultured mouse collicular and striatal neurons and the reagent proved non-toxic and produced comparable transfection efficiencies. The time point of transfection following plating the cells was important: Greatest transfection efficiency into granule cells (~2%) was seen when transfection occurred one day (~24 hours) after plating. The number of GFP-expressing cells increased for the first three days after transfection and then remained stable for two weeks. (1478)

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J. Biol. Chem. 274, 29720-29725. Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells. 1999

Halim, A.-B., LeGros, L., Geller, A., Kotb, M.

Notes: Subunits of the cDNA for methionine adenosyltransferase were amplified from a plasmid using Taq DNA Polymerase and directly cloned into the pTARGET™ Mammalian Expression T-Vector. The sequence of the cloned inserts were confirmed with the fmol® DNA Cycle Sequencing System. The constructs were transiently transfected into COS-1 cells with the TransFast™ Reagent at a 1:1 ratio. Excellent detail is provided for the transfection. (1094)

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J. Biol. Chem. 273, 26269-26272. BEGAIN (brain-enriched guanylate kinase-associated protein), a novel neuronal PSD-95/SAP90-binding protein. 1998

Deguchi, M., Hata, Y., Takeuchi, M., Ide, B., Hirao, K., Yao, I., Irie, M., Toyoda, A., Takai, Y.

Notes: CHO cells stably transfected with the PSD-95/SAP90 protein were generated using the pCI-Neo Mammalian Expression Vector. These stably transfected cells or standard CHO cells were transiently transfected with various eukaryotic expression constructs including some in the pCI-neo Vector. Transient transfection of the CHO cells was performed with the TransFast™ Reagent. (1267)

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Immunity 9, 787-796. Engagement of GPI-linked CD48 contributes to TCR signals and cytoskeletal reorganization: A role for lipid rafts in T cell activation. 1998

Moran, M., Miceli, M.C.

Notes: The TransFast™ Reagent was used to stably transfect L cells with a G418-resistant expression vector. No details of the transfection are provided. (0658)

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Proc. Natl. Acad. Sci. USA 95, 13513-13518. The lipid phosphate activity of PTEN is critical for its tumor supressor function. 1998

Myers, M.P., Pass, I., Batty, I.H., Van der Kaay, J., Stolarov, J.P., Hemmings, B.A., Wigler, M.H., Downes, C.P., Tonks, N.K.

Notes: The TransFast™ Reagent was used to transfect LnCaP cells using a 1:1 lipid:DNA ratio. Cells were cotransfected with a GFP vector for selection of positive cells 12 h post transfection. (0638)

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