Targeted modification of a human beta-globin locus BAC clone using GET Recombination and an I-Scei counterselection cassette.
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 beta-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.
Jamsai
Duangporn
D
CAGT Research Group, The Murdoch Childrens Research Institute, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia.
Orford
Michael
M
Nefedov
Mikhail
M
Fucharoen
Suthat
S
Williamson
Robert
R
Ioannou
Panayiotis A
PA
D001483:Base Sequence; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D005914:Globins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins
D029701:Saccharomyces cerevisiae Proteins; D005914:Globins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific