An adenoviral vector for probing promoter activity in primary immune cells.
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.
Division of Immunobiology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
Pulak
P
Tripathi
Rajat
R
Madan
Claire
C
Chougnet
Senad
S
Divanovic
Xiaojing
X
Ma
Larry M
LM
Wahl
Thomas
T
Gajewski
Christopher L
CL
Karp
David A
DA
Hildeman
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003713:Dendritic Cells / Q000276:immunology / Q000502:physiology; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology; D053759:Interleukin-23; D053760:Interleukin-23 Subunit p19; D007378:Interleukins / Q000235:genetics / Q000276:immunology; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics; D008264:Macrophages / Q000276:immunology / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D050778:NFATC Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000276:immunology / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology / Q000502:physiology* / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods*
C508840:IL23A protein, human; C508841:Il23a protein, mouse; D007376:Interleukin-2; D053759:Interleukin-23; D053760:Interleukin-23 Subunit p19; D007378:Interleukins; D050778:NFATC Transcription Factors; D018664:Interleukin-12; D008156:Luciferases