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An adenoviral vector for probing promoter activity in primary immune cells.

TitleAn adenoviral vector for probing promoter activity in primary immune cells.
Publication TypeJournal Article
Year of Publication2006
AuthorsTripathi P, Madan R, Chougnet C, Divanovic S, Ma X, Wahl LM, Gajewski T, Karp CL, Hildeman DA
JournalJ Immunol Methods
Volume311
Issue1-2
Pagination19-30
Date Published2006 Apr 20
ISSN0022-1759
KeywordsAdenoviridae, Animals, Dendritic Cells, Flow Cytometry, Gene Expression Regulation, Genetic Vectors, Humans, Interleukin-12, Interleukin-2, Interleukin-23, Interleukin-23 Subunit p19, Interleukins, Jurkat Cells, Luciferases, Macrophages, Mice, Mice, Transgenic, NFATC Transcription Factors, Promoter Regions, Genetic, Regulatory Sequences, Nucleic Acid, T-Lymphocytes, Transduction, Genetic
Abstract

Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter-reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells.

DOI10.1016/j.jim.2006.01.009
Alternate JournalJ Immunol Methods
PubMed ID16563424
PubMed Central IDPMC2964867
Grant ListR01 CA100223 / CA / NCI NIH HHS / United States
AI056927 / AI / NIAID NIH HHS / United States
R01 CA100223-01A1 / CA / NCI NIH HHS / United States
R01 AI057753 / AI / NIAID NIH HHS / United States
R01 DK056415 / DK / NIDDK NIH HHS / United States
DK56415 / DK / NIDDK NIH HHS / United States
R01 AI056927 / AI / NIAID NIH HHS / United States
R56 AI057753 / AI / NIAID NIH HHS / United States
R01 NS039435 / NS / NINDS NIH HHS / United States
NS39435 / NS / NINDS NIH HHS / United States
AI057753 / AI / NIAID NIH HHS / United States

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