Insertion of modifications in the beta-globin locus using GET recombination with single-stranded oligonucleotides and denatured PCR fragments.
We describe the use of the GET recombination system with oligonucleotides or single-stranded polymerase chain reaction (PCR) fragments to insert modifications in the human beta-globin locus without counterselection. The method involves recombination between oligonucleotides or denatured PCR fragments and homologous sequences in the beta-globin gene in a clone of 205-kb bacterial artificial chromosome (BAC), based on the inducible expression of the recE, recT, and gam genes. In this method, oligonucleotides or denatured PCR fragments are electroporated directly into cells carrying both the globin BAC and the pGETrec plasmid, after induction of the GET recombination system. Recombinant BAC clones are identified by PCR, using allele-specific amplification for the mutated sequences. We have used this approach to insert a unique restriction site as well as a common thalassemia mutation (stop codon 39, C-->T) into the human beta-globin locus. We have observed the frequency of recombinant clones to be as high as 1 in 100-200 clones. Therefore, this approach provides a simple and efficient method for introducing point mutations and other fine modifications into BACs, and should greatly facilitate the use of BACs for functional studies and therapeutic applications.
Jamsai
Duangporn
D
CAGT Research Group, The Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Victoria 3052, Australia.
Orford
Michael
M
Fucharoen
Suthat
S
Williamson
Robert
R
Ioannou
Panayiotis A
PA
D001483:Base Sequence; D002478:Cells, Cultured; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics; D011995:Recombination, Genetic; D013789:Thalassemia / Q000235:genetics; D014184:Transplantation, Homologous / Q000379:methods*
D003062:Codon; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011994:Recombinant Proteins; D005914:Globins