Generation of Lentiviral Constructs for NF-κB Assay

Human IL-10 was amplified from pUbC-hIL-10 (provided by Prof. David Dean, University of Rochester Medical Center) and ligated into the viral plasmid backbone, HIV-CSCG (provided by Prof. David Schaffer, University of California, Berkeley) using NheI and XhoI (New England Biosciences, Ipswich, MA). Lentivirus reporter constructs containing enhancer elements bound by NF-κB and encoding firefly luciferase were employed to assay transcription factor (TF) activity along with a negative control construct containing only a TATA box promoter (Panomics, Freemont, CA) by live imaging (Weiss et al., 2012 (link)). Ligated plasmids were transformed into DH5α cells (Invitrogen). Plasmids were purified using Qiagen reagents and stored in Tris–EDTA buffer (Qiagen, Venlo, Limburg, Netherlands) at −20°C.

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Boehler R.M., Kuo R., Shin S., Goodman A.G., Pilecki M.A., Leonard J.N, & Shea L.D. (2014). Lentivirus Delivery of IL-10 to Promote and Sustain Macrophage Polarization Towards an Anti-Inflammatory Phenotype. Biotechnology and bioengineering, 111(6), 1210-1221.

Publication 2014

DH5α cells Tris–EDTA buffer

Assay Backbone Cells Edta Enhancer elements Firefly luciferase Human Il 10 Lentivirus Nf κb Plasmids Tata box Transcription factor Tris buffer

Corresponding Organization : Northwestern University

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Variable analysis

independent variables

Lentivirus reporter constructs containing enhancer elements bound by NF-κB and encoding firefly luciferase
Negative control construct containing only a TATA box promoter

dependent variables

Transcription factor (TF) activity

control variables

Plasmids were transformed into DH5α cells
Plasmids were purified using Qiagen reagents and stored in Tris–EDTA buffer at −20°C

controls

Negative control construct containing only a TATA box promoter

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