Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (a) Nucleofection with pmaxGFP. MSCs were nucleofected with pmaxGFP and imaged at 6 and 24 hours post nucleofection. Brightfield and fluorescent images were obtained using a Nikon Eclipse TS2 microscope at 20x magnification, 100μm scale. (b) Schematic representation of the pVITRO2-Luc2 and pVITRO2-Luc2/CDUPRT mammalian expression plasmids. (c) Antibiotic sensitivity assay. Native MSCs were grown in the presence of a 2-fold serial dilution of Hygromycin B (0–500μg/ml) for 14 days. (d) Nucleofection with bioluminescent plasmids. MSCs were nucleofected with pVITRO2- Luc2 and pVITRO2- Luc2/CDUPRT , and selected for with Hygromycin B for two weeks. Images were acquired on a Leica DM Microscope at 10x magnification, 100μm scale. (e) BLI in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 over a linear cell concentration range. The cells were incubated with 1:1 D-luciferin (300μg/ml) and imaged on the IVIS Lumina II according to the in vitro BLI acquisition settings. The images are represented at t = 30min. Lane 1: D-PBS, Lane 2–10: Nucleofected MSC vs control MSC. (f) Analysis of luciferase activity in MSC- Luc2 , MSC- Luc2/CDUPRT #1 and MSC- Luc2/CDUPRT #2 . Luminescence was captured at multiple time-points following incubation with D-luciferin was analyzed using GraphPad Prism 7. Data are represented as the mean average radiance ± SDs of triplicates.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: Microscopy, Expressing, Sensitive Assay, Serial Dilution, Concentration Assay, Incubation, In Vitro, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: Luciferase-based reporting of suicide gene activity in murine mesenchymal stem cells

doi: 10.1371/journal.pone.0220013

Figure Lengend Snippet: (A) In vitro BLI of CDUPRT activity. BLI was assessed following the addition of 5-FC and 5-FU to MSC- Luc2 (A-D) and MSC- Luc2/CDUPRT #1 (E-H). For 5-FC BLI data: Lanes 1–12 contains 2-fold serial dilutions of 5-FC from 0–2000μg/ml. For 5-FU BLI data: Lanes 1–7 contains 10-fold serial dilutions of 5-FU from 0–1000μg/ml. (B) Analysis of BLI data. Data are represented as the mean average radiance ± SEMs (n = 3). Baseline luminescence is indicated by a dotted-line. A two-way ANOVA and Sidak’s post-hoc were performed, p<0.05.

Article Snippet: Briefly, the luciferase reporter gene Luc2 ( Photinus pyralis ), encoded within the vector pGL4.20 ( Luc2 /Puro) (Promega, USA), was digested with the restriction enzymes EcoRV-HF and BamHI-HF (New England Biolabs, USA), and ligated into the mammalian dual expression plasmid pVITRO2-hygro-mcs (InvivoGen, USA), to generate the plasmid pVITRO2- Luc2 .

Techniques: In Vitro, Activity Assay