Luciferase silencing

Luciferase gene expression is common as well as green fluorescent protein expression. These are some standard examples used to evaluate gene silencing. 

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...

Figure 12 In vitro evaluation of the lipidoid library for sIRNA delivery, (a) Percent reduction of firefly luciferase expression in HeLa cells with lipidoid/ siRNA formulations. The data are split into five groupings spanning 0-100% luciferase reduction for ease of analysis, (b) Optimal knockdown levels in HeLa determined by screening at four different lipidoid/siRNA ratios, (c-e) Dose response silencing in HeLa (c), HepG2 (d), and primary macrophage (e) cells. Reprinted from Akinc, A. Zumbuehl, A. Goldberg, M. et at. Nat. Biotechnol. 2008, 26 (5), 561-569, with permission from the Nature Publishing Group.

The reporter construct used to establish stable reporter cell lines is shown in Fig. 1. The construct consists of a reporter gene, in this case lacZ or luciferase, driven by a minimal promoter containing a TATA motif and a RARE. The RARE/TATA promoter is derived from the human p-retinoic acid receptor gene (8) and functions as an inducible enhancer that responds to all known RAR subtypes (19). Immediumtely 5 to the RARE is a silencer cassette... 

The function of the ER domains is to silence the enzymatic activity of the luciferase. The construct was then stably transfected into human glioma (D54) cells and the cells constitutively expressing ER-DEVD-Luc-DEVD-ER were grown in cell culture and subcutaneous tumors were implanted in athymic... 

Fig. 12 Gene-silencing effect of chemically synthesized pre-nuRNA. Left-. Effect of pre-nuRNA on expression of luciferase target gene. G3T-hi cells were transfected with pHOXB-Luc reporter plasmid, and effector RNA (100 nM) and reporter assays performed 48 h after transfection. Each value shown is the average of three independent experiments, and standard deviations are indicated as error bars. Right. Sense/antisense suppression activity ratios. G3T-hi cells were transfected with pHOXB-Luc or pHOXB-Luc-antisense reporter plasmid and effector RNA (30 nM), and reporter assays performed 48 h after transfection. The results are presented as the ratio of the average percent suppression of luciferase-containing sense and antisense target by the 110-nt pre-miRNA or the 22-nt miRNA duplex [111]...

Dinh TT et al (2013) Generation of a luciferase-based reporter for CHH and CG DNA methylation in Arabidopsis thaliana. Silence 4 1... 

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