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.
Tripathi
Pulak
P
Division of Immunobiology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA.
Madan
Rajat
R
Chougnet
Claire
C
Divanovic
Senad
S
Ma
Xiaojing
X
Wahl
Larry M
LM
Gajewski
Thomas
T
Karp
Christopher L
CL
Hildeman
David A
DA
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