Duangporn JamsaiMichael OrfordMikhail NefedovSuthat FucharoenRobert WilliamsonPanayiotis A. IoannouUniversity of MelbourneMahidol UniversityCyprus Institute of Neurology and GeneticsChildren's Hospital Oakland Research Institute2018-07-242018-07-242003-07-01Genomics. Vol.82, No.1 (2003), 68-77088875432-s2.0-0037603297https://repository.li.mahidol.ac.th/handle/20.500.14594/20714There 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/S0888-7543(03)00100-9