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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)

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)

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)

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)

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)

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)

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)

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)

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)

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: 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)

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)

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)

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 denditric 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)

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)

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)

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)

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)

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)