XB-IMG-124550
Xenbase Image ID: 124550
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Figure 5. Analysis of a promoter-less bicistronic construct containing the MMTV 5â²-UTR sequence. (A) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ÎEMCV (lane 3) or dl 5â²UTR-MMTV was removed to generate the equivalent promoter-less (ÎSV40) vectors. (B) NMuMG cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) together with the pcDNA3.1 lacZ (50 ng) plasmid. Total DNA was extracted from transfected NMuMG cells and the presence of the transfected plasmids was confirmed by PCR. Plasmids (100 ng) dl ÎEMCV (lane 3) or dl 5â²UTR-MMTV (lane 2) were used as amplification controls. (C) Total RNA was extracted from transfected NMuMG cells and the presence of transcripts for the dl ÎEMCV (lane 3), the dl 5â²UTR-MMTV, the Î SV40-dl ÎEMCV and Î SV40-dl 5â²UTR-MMTV was evaluated by a one-step RT-PCR designed to detect the bicistronic RNA (depicted in Figure 3B). In vitro transcribed RNA (100 ng) generated from plasmids dl ÎEMCV (lane 3) or dl 5â²UTR-MMTV (lane 2) were used as amplification controls. The presence of template RNA was confirmed in parallel by loading the total RNA (10 µg) used in the RT-PCR onto a 0.7% denaturing agarose gel. (D) NMuMG cell were co-transfected with either the SV40 or Î SV40 version of dl ÎEMCV (200 ng) or dl 5â²UTR-MMTV (200 ng) plasmids together with the pcDNA3.1 lacZ (50 ng) plasmid. Cells were processed, and β-galactosidase and total proteins were quantified. RLuc and FLuc activities were measured, and data are presented as [RLuc/(total protein) à (β-galactosidase)] (left panel) and [FLuc/(total protein) à (β-galactosidase)] (right panel). Image published in: Vallejos M et al. (2010) © The Author(s) 2009. Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View |