Premium
Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene
Author(s) -
Ikeno Masashi,
Inagaki Hidehito,
Nagata Keiko,
Morita Miwa,
Ichinose Hiroshi,
Okazaki Tuneko
Publication year - 2002
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1046/j.1365-2443.2002.00580.x
Subject(s) - biology , gtp cyclohydrolase i , gene , microbiology and biotechnology , guanosine , tetrahydrobiopterin , enhancer , transfection , gene expression , biochemistry , enzyme , nitric oxide synthase
Background Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing α‐satellite DNA with CENP‐B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co‐factor of aromatic amino acid hydroxylases and nitric oxide synthase. Results: We constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co‐transfection with the α‐satellite DNA‐containing BAC to a human fibroblast cell line. Two cell lines carrying a HAC with GCH1 genes were obtained. Both HACs were composed of multiple copies of precursor BACs and were maintained stably in human and mouse cell lines. The GCH1 activities of the HAC‐carrying human fibroblast cell lines were elevated but still highly sensitive to IFN‐γ induction, mimicking the response of the gene expression from the authentic chromosomal genes. Conclusion: These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.