Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.
GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.
Orford
M
M
CAGT Research Group, The Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, Melbourne, Victoria 3052, Australia.
Nefedov
M
M
Vadolas
J
J
Zaibak
F
F
Williamson
R
R
Ioannou
P A
PA
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter; D016680:Genome, Bacterial; D005914:Globins / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D007612:Kanamycin; D008164:Luminescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007612:Kanamycin; D005914:Globins