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Nucleic Acids Research, 2000, Vol. 28, No. 18 3605-3614
© 2000 Oxford University Press

Stable expression and cell-specific chromatin structure of human {alpha}1-antitrypsin cosmid transgenes in rat hepatoma cells

Pierre Rollini, Lianjun Xu and R. E. K. Fournier*

Division of Basic Sciences, A2-025, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Seattle, WA 98109-1024, USA

The human gene encoding {alpha}1-antitrypsin ({alpha}1AT, gene symbol PI) resides in a cluster of serine protease inhibitor (serpin) genes on chromosome 14q32.1. {alpha}1AT is highly expressed in the liver and in cultured hepatoma cells. We recently reported the chromatin structure of a >100 kb region around the gene, as defined by DNase I-hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Transfer of human chromosome 14 by microcell fusion from non-expressing fibroblasts to rat hepatoma cells resulted in activation of {alpha}1AT transcription and chromatin reorganization of the entire region. In the present study, we stably introduced cosmids containing {alpha}1AT with various amounts of flanking sequence and a linked neo selectable marker into rat hepatoma cells. All single-copy transfectants with >14 kb of 5' flanking sequence expressed wild-type levels of {alpha}1AT mRNA in a position-independent manner. In contrast, expression of transgenes containing only ~1.5–4 kb of flanking sequence was highly variable. Long-term culture of transfectant clones in the absence of selection resulted in gradual loss of neo expression, but expression of the linked {alpha}1AT gene remained constant. DHS mapping of cosmid transgenes integrated at ectopic sites revealed a hepatoma-specific chromatin structure in each transfectant clone. The implications of these findings are discussed.

* To whom correspondence should be addressed. Tel: +1 206 667 5217; Fax: +1 206 667 6522; Email: kfournie@fhcrc.org


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 Mol. Cell. Biol.Home page
M. D. Marsden and R. E. K. Fournier
Chromosomal Elements Regulate Gene Activity and Chromatin Structure of the Human Serpin Gene Cluster at 14q32.1
Mol. Cell. Biol., May 15, 2003; 23(10): 3516 - 3526.
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