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Title: Targeted modification of a human β-globin locus BAC clone using GET Recombination and an I-SceI counterselection cassette
Authors: Duangporn Jamsai
Michael Orford
Mikhail Nefedov
Suthat Fucharoen
Robert Williamson
Panayiotis A. Ioannou
University of Melbourne
Mahidol University
Cyprus Institute of Neurology and Genetics
Children's Hospital Oakland Research Institute
Keywords: Biochemistry, Genetics and Molecular Biology
Issue Date: 1-Jul-2003
Citation: Genomics. Vol.82, No.1 (2003), 68-77
Abstract: There is a need for better approaches to allow precise engineering of large genomic BAC DNA fragments, to facilitate the use of intact genomic loci for therapeutic and biotechnology applications. We report an efficient method to insert any modification in any genomic locus, using a human β-globin locus BAC clone as a model system. The modifications can range from single base changes to large insertions or deletions and leave no operational sequences. A counterselection cassette, consisting of an inducible I-SceI gene, its recognition site, and an antibiotic resistance gene, is inserted into the targeted region using GET Recombination. A PCR fragment carrying the modification but no selectable marker replaces the counterselection cassette in a second round of GET Recombination. The unique I-SceI site in the counterselection cassette is cut by I-SceI endonuclease, strongly selecting against nonrecombinant clones and yielding up to 30% correct recombinants. © 2003 Elsevier Science (USA). All rights reserved.
ISSN: 08887543
Appears in Collections:Scopus 2001-2005

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