A New Regulatory Region of the IL-2 Locus That Confers Position-Independent Transgene Expression
Abstract
Although the promoter/enhancer of the IL-2 gene mediates inducible reporter gene expression in vitro, it cannot drive consistent expression in transgenic mice. The location and existence of any regulatory elements that could open the IL-2 locus in vivo have remained unknown, preventing analysis of IL-2 regulation in developmental contexts. In this study, we report the identification of such a regulatory region, marked by novel DNase-hypersensitive sites upstream of the murine IL-2 promoter in unstimulated and stimulated T cells. Inclusion of most of these sites in an 8.4-kb IL-2 promoter green fluorescent protein transgene gives locus control region-like activity. Expression is efficient, tissue specific, and position independent. This transgene is expressed not only in peripheral T cells, but also in immature thymocytes and thymocytes undergoing positive selection, in agreement with endogenous IL-2 expression. In contrast, a 2-kb promoter green fluorescent protein transgene, lacking the new hypersensitive sites, is expressed in only a few founder lines, and expression is dysregulated in CD8+ cells. Thus, the 6.4 kb of additional upstream IL-2 sequence contains regulatory elements that provide integration site independence and differential regulation of transgene expression in CD8 vs CD4 cells.
Additional Information
© 2001 American Association of Immunologists. We thank Stephen Hedrick for kindly providing the plasmid containing the human β-globin 3′ splicing and poly(A) addition sites and Michael Nishimura for the melanoma cell line MCA102. We gratefully acknowledge help from Robert Chen for screening the genomic library for the IL-2 promoter clone and to Xiao Sun for sequencing assistance. Shelley Diamond and Pat Koen of the Flow Cytometry Core Facility provided invaluable help with flow cytometry and cell sorting. We also thank Shirley Pease, Xin Yu, Bruce Kennedy, and Alba Granados of the Caltech Transgenic and Knockout Core Facility for the generation and maintenance of the transgenic mice. All primers were made at the Caltech Biopolymer Synthesis Facility, and sequencing was performed at the Caltech DNA Sequencing Core Facility. This work was supported by National Institutes of Health Grant AG13108 and the Stowers Institute for Medical Research.Additional details
- Eprint ID
- 74223
- Resolver ID
- CaltechAUTHORS:20170213-074814967
- AG13108
- NIH
- Stowers Institute for Medical Research
- Created
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2017-02-13Created from EPrint's datestamp field
- Updated
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2021-11-11Created from EPrint's last_modified field