Insertion of common mutations into the human β-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette

Abstract

A large number of mutations have been described in the human β-globin locus causing thalassemia or various hemoglobinopathies. However, only a very limited number of these mutations have been studied in animal model systems in the context of the human β-globin locus. We report here the use of the GET Recombination system with an EcoRI/KanR counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG→AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human β-globin locus. The counterselection cassette was first inserted into the target sequence in the β-globin gene, and then a PCR fragment carrying the re..