Luciferase reporter plasmids

Fig. 3. (A) SK-N-SH neuronal cells were cotransfected with Bcl-xL or Bcl-xL AkB luciferase reporter plasmids together with pSG-c-Rel and pSG-RelA, or a combination ofpSG-p50, pSG-RelA, andpSG-c-Rel expression plasmids. Control cells were transfected with empty pSG5 vector. Twenty-four hours later the luciferase activity was measured. Mutation of NF- B binding site blocked the luciferase expression. The c-Rel-containing dimers, p50/c-Rel and RelA/c-Rel, but not p50/RelA complex, were able to activate Bcl-xL promoter. Values are mean S.E.M. of three experiments run in triplicate ( /> < 0.05 vs. the corresponding control value). (B) OGD-induced NF-acB activation promotes Bim but not Bcl-xL transcription. Primary cortical neurons were transfected with Bim or Bcl-xL luciferase reporter plasmid or with Bim and Bcl-xL AkB luciferase reporter plasmids and then exposed to OGD. Four hours later, luciferase activity was measured. The OGD exposure significantly induced Bim and decreased Bcl-xL promoter activity. Mutation of NF-kB binding sites reduced the luciferase expression. Values are mean S.E.M. of three experiments run in triplicate p < 0.05 vs. the corresponding control value p < 0.05 vs. corresponding wild-type luciferase reporter plasmid). For methods and details, see Samico et al. (2009).

Plasmid DNA solution prepare DNA solution e.g. luciferase reporter plasmid or eGFP plasmid, at a concentration of 12 pg DNA/ml by dilution of the stock solution with a serum- and supplement-free medium (e.g., RPMl 1640). 

Luciferase reporter plasmid p55pCMV-lVS-luc +containing the firefly luciferase cDNA under the control of the cytomegalovirus (CMV) promoter. 

Most recently, low-intensity ultrasound has been used to enhance gene transfection by liposome.139 In this study, ultrasound exposure (1 MHz, 0.4 W/cm2) for 60 seconds enhanced transfection of naked or liposome-complexed luciferase reporter plasmid into cultured porcine vascular smooth muscle and endothelial cells. These results with liposome-complexed reporter plasmid are similar to those of Unger et al., who showed that relatively low levels of ultrasound energy (0.5 W/ cm2) enhanced gene expression.140 In these two studies, ultrasound exposure did... 

MG-63-cells cotransfected with a luciferase reporter plasmid (pGVB2 vector) inserted with a rat 25-hydroxyvitamin D3-24-hydroxylase gene promotor (-291/+3) including two VDREs as an internal control. 

To investigate if camosol specifically inhibited activation of NF-kB, gel mobility shift assay was performed to analyze NF-kB DNA binding activity. As shown in a previous study (25), the induction of specific NF-kB DNA binding activity by LPS was inhibited by camosol. The specificity of binding was examined by competition with the addition of excess consensus unlabeled oligonucleotide. In an additional study, transient transfection with a NF-kB-dependent luciferase reporter plasmid was done to confirm whether camosol inhibited NF-kB binding activity in LPS-induced macrophages. The results indicated that camosol inhibited LPS-induced NF-kB transcriptional activity. 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

This assay has been thoroughly reviewed [34] and is outlined in Fig. 5. In brief, a cell is transfected with a reporter plasmid consisting of a GAL4 response element upstream from luciferase. NRs are produced as chimeras consisting of the GAIA... 

Figure 1 The principles and variant parameters of lipofection. (i) Preparation of a lipofection reagent cationic liposomes were prepared from cationic lipids and helper (if required), (ii) Formation of positively charged lipoplexes by addition of DNA (e.g., reporter plasmid carrying the firefly luciferase gene) to the cationic liposomes, (iii) Transfection (lipofection) by incubation cells with the preformed lipoplexes. The efficiency of gene transfer (lipofection efficiency) can be determined from reporter gene amount or activity (e.g., luciferase activity). Most of the steps of a lipofection experiment can be varied and optimized (grey spots).

Plasmid DNA used contained the luciferase reporter gene (for in vitro experiments) including the CMV sequence. Plasmids present the normalized criteria of quality endotoxin level <20 EU/mg, supercoiled DNA >90%, E. coli-derived DNA contaminant <5%, RNA contaminant <5%, and protein contamination <1%. Such a plasmid preparation has been described (16). 

Fig. 2. Transfection of the cationic and anionic formulations using a plasmid encoding for the luciferase reporter gene in presence and absence of bafilomycin. The Y axis represents the level of luciferase expression per protein (p.g), mean values+SD. The background level of the untreated cells was removed for each sample...

ReProGlo assay ReProGlo Luciferase activity (BMC, benchmark concentration) ofTcf/ Lef-promoter-driven reporter plasmid in mouse embryonic stem cells (Wnt signaling) Embryonic development... 

Enhancer-dependent expression is observed following transplantation of an injected pronucleus into a two-cell blastomere (Henery et al., 1995) (Figure 4). The first mitosis—and concomitant loss of the plasmid from the injected pronucleus—was circumvented by the transplantation procedure and hence cannot account for the enhancer requirement observed in the two-cell embryo. The luciferase reporter gene is driven by the / promoter, which has two Spl binding sites (termed the proximal and distal sites)—a CAAT box binding site that binds CTF and a TATA box that binds TBP. Analysis of the expression of a set of linker scanning mutations that inactivated each of these sites without altering the distances between each of the promoter elements revealed that each mutation had the same relative effect on tk... 

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