HeLa, luciferase

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. 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

Figure 6.2 Critical parameters of the miR/mRNA co-transfection method. (A) Titration of mRNA amount. HeLa cells were transfected with increasing amounts of cap tail R-luc-4 sites mRNA and a fixed amount of firefly (F-luc) mRNA. R-luc expression (luciferase activity) was measured 5 h after transfection. (B) Titration of miCXCR4 concentration. HeLa cells were transfected with cap tail R-luc-4 sites mRNA, F-luc mRNA, and varying concentrations of miCXCR4. Luciferase activity was measured 16 h after transfection and fold-repression by the miR was calculated as in Fig. 6.1D (C) Time-course of miR-mediated repression. HeLa cells were co-transfected with cap tail R-luc-4 sites and F-luc (control) mRNAs, either with or without miCXCR4, and harvested at different time points. Repression was calculated as detailed in Fig. 6.1 and plotted against time (mRNA transfection data series depicted by the circles). Analogous plasmid DNA transfections are shown for reference (pDNA, diamonds). Averaged results from several experiments are shown with standard deviation. Data were previously published (Humphreys etal., 2005). Copyright PNAS, reprinted with permission.

An example of miR-dependent deadenylation of mRNA measured by this method is shown in Fig. 6.3C. In this case, HeLa cells (which express let-7) were transfected with a pDNA R-luc construct encoding three let-7 binding sites in the 3 UTR (3 X bulge), or with control constructs encoding either no sites (plasmid) or three mutated let-7 sites (3 x bulge mut) (constructs described in Pillai et al, 2005). Cells were harvested 24 h after transfection, RNA was purified for the PAT assay, and luciferase activity was measured from cell lysates. As reported previously, the presence of functional let-7 target sites results in specific repression of luciferase expression with very minor effects on mRNA stability (Pillai et al., 2005). The experiment in Fig. 6.3C demonstrates that the let-7 targeted reporter mRNA is selectively deadenylated. 

Fig. l.A sample plate from a screen to identify small molecules that upregulate the expression of a cyclin-luciferase reporter protein. HeLa cells were infected with an adenovirus expressing cyclin-luciferase, as described in the methods section. The graph shows the activity of all wells from a single 384-well plate. Several compounds that upregulate cyclin-luciferase expression were identified. 

Fig. 2. TransIT-LTl-mediated transfection of HeLa cells at varying cell densities HeLa cells were plated in 12-well plates and transfected in parallel at 50% and 90% confluency. TransIT-LTl reagent transfections were performed in duplicate using a luciferase expression vector (pCI-luc) and 3 iL reagent per well, shows the importance of plating cells at optimal density for transfection. Twenty-four hours post-transfection, cells were harvested and assayed for luciferase activity. Visual confluence (line graph) was measured under the microscope at harvest. The data represent the average luciferase activity (Relative Light Units - RLUs in Millions) from three experiments performed on different days.

HeLa pLuc705 cells are stably transfected by a luciferase construction allowing the quantitative assessment of PNA-peptide conjugates nuclear delivery and biological activity (Fig. 1). Cells should not be passaged more than ten times and should be checked routinely (—one to two times per month) for the absence of mycoplasma contamination... 

We reported that greater transfection efficiency in medium with serum was obtained in human cervical carcinoma HeLa cells, using (I) DC-Chol/DOPE liposomes (molar ratio, 1 2) than liposomes (1 1 or 3 2), (2) a modified ethanol injection (MEI) method to prepare liposomes than the dry-film method (13, 14), and (3) a dilution method to form lipoplex than direct mixing. The physicochemical properties of liposomes and lipoplexes can be examined by measuring particle size. Transfection efficiency was evaluated by using plasmid DNA encoding luciferase gene and the cells. 

Formulations containing pH labile cationic lipids 2 and 4 were evaluated in vitro in transfection experiments with Hela cells with three different charge ratios (1.5, 6.0, and 10), using plasmid DNA containing the luciferase (Luc) reporter gene under the cytomegalovirus (CMV) promoter. 

Fig. 1. Effect of vortex speed (2,500 rpm for 10 min) on the luciferase gene siiencing efficiency of a siRNA-LipoTrusf lipoplex in HeLa cells. The amount of cationic lipid was 9.6 aM. The siRNA doses correspond to the cationic Npid+/siRNA- charge ratio of 30.45, 15.24,7.62,3.81,1.90 and 0.95, respectively. LipoTrusf is constituted of dioleoylphos-phatidylethanolamine, cholesterol and the cationic lipid 0,0 -ditetradecanoyl-/V-(a-trimethyl ammonioacetyl) diethanolamine chloride (DC-6-14) in the molar ratio of 0.75/0.75/1.00. For this cationic liposome, an expressive gene silencing effect in vitro was obtained at lower siRNA dose with application of a higher vortex-mixing during complex formation...

Fig. 13 Panel for triblock copolymer PEG-PAsp(MPA)-PLL system, a Chemical structure of PEG-PAsp(MPA)-PLL. b Schematic illustration hypothesizing a three-layered micelle formed from the triblock copolymer and pDNA with spatially regulated structure, c In vitro transfection of the luciferase gene to HeLa cells by the micelles from di- or triblock copolymers and polyplex with PEI. The micelles were prepared at a Lys/nucleotide ratio of 2. HeLa cells were incubated with each micelle in a mediiun containing 10% serinn for 24 h, followed by additional 24 h incnbation withont micelles, d The effects of HCQ and NR on the transfection efficiency of the micelles and polyplex. The PEI polyplex was prepared at a N/P ratio of 10. (Fig. 13d Reprinted with permission from [116])...

Konopka, K., Harrison, G. S., Felqner, P. L., and Duzgunes, N. (1997) Cationic liposome-mediated expression of HIV-regulated luciferase and diphtheria toxin a genes in HeLa cells infected with or expressing HIV. Biochim. Biophys. Acta 1356, 185-197. 

Cytosolic ATP dynamics of HeLa cell during apoptosis was measured using a luminometer (Fig.l). Total amount of ATP increased within 20 to 120 min and decreased gradually within 10 h via three types of apoptotic induction (STS, FCCP and CHX). As CHX is an inhibitor of protein synthesis, ATP elevation after apoptotic induction is not due to elevation of luciferase synthesis. On the other hand, ATP elevation is suppressed by glucose-free medium and 2-deoxy-D-glucose. It leads to a hypothesis that glycolysis pathway contributes to an elevation of cytosolic ATP.5 This hypothesis is supported by the result using FCCP, an inhibitor of mitochondrial ATP production. 

Ca2+ imaging. HeLa cell co-transfected apoobelin and c-fos promoter constructs were incubated in Dulbecco s modified Eagle s medium containing coelenterazine (Renilla luciferase assay system, Promega) for 4 h to reconstruct obelin. The cell was stimulated by 500 (xM ATP, and the luminescence image captured on a luminescence microscope (Luminoview, LV 200, Olympus) equipped with iXon EM-CCD camera (Andor). Binning of the CCD was 2x2, and the exposure time was 25 s with 30 s interval. The cell was re-stimulated by 10 pM ionomysin at 20 min after ATP stimulation. 

To test for promoter activity, HeLa cells were transfected with pRSV-B-gal and either p5 2ANOS-L or p5 2BNOS-L, using calcium phosphate-mediated gene transfer (Chen and Okayama, 1987). After 36 hr, cell extracts were prepared and assayed for jS-ga/actosidrtse and luciferase activities. The luciferase activities were normalized to 8-galactosidase activities so that... 

Recent data demonstrate that the activities of certain transcription factors are regulated during the cell cycle (Dowdy etal., 1993). It is therefore possible that aberrant expression of NOSl-luciferase fusion genes in HeLa cells reflects the mitotic activity of the cell culture system. 

While transient expression of NOSl-luciferase fusion genes in HeLa cells remains enigmatic, CNS-specific expression of a NOSl-lacZ fusion... 

Zacharewski et al. (1998) found that di- -butyl phthalate induced reporter gene (luciferase) activity in MCF-7 human breast cancer cells, but not HeLa human cervical carcinoma cells, transfected with human estrogen receptor, although approximately 3,000-fold less potent than 17p-estradiol. Additionally, Zacharewski et al. (1998) found that di- -butyl phthalate was capable of supporting a very modest amount... 

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