title: Transgene copy number-dependent rescue of murine beta-globin knockout mice carrying a 183 kb human beta-globin BAC genomic fragment.
abstract: We report the generation and characterisation of the first transgenic mice exclusively expressing normal human beta-globin ((hu)beta-globin) from a 183 kb genomic fragment. Four independent lines were generated, each containing 2-6 copies of the (hu)beta-globin locus at a single integration site. Steady state levels of (hu)beta-globin protein were dependent on transgene copy number, but independent of the site of integration. Hemizygosity for the transgene on a heterozygous knockout background ((hu)beta(+/0), (mu)beta(th-3/+)) complemented fully the hematological abnormalities associated with the heterozygous knockout mutation in all four lines. Importantly, the rescue of the embryonic lethal phenotype that is characteristic of homozygosity for the knockout mutation was also demonstrated in two transgenic lines that were homozygous for two copies of the (hu)beta-globin locus, and in one transgenic line, which was hemizygous for six copies of the (hu)beta-globin locus. Our results illustrate the importance of transgene copy number determination and of the hemizygosity/homozygosity status in phenotypic complementation studies of transgenic mice containing large heterologous transgenes. Transgenic mouse colonies with 100% (hu)beta-globin production from the intact (hu)beta-globin locus have been established and will be invaluable in comparative and gene therapy studies with mouse models containing specific beta-thalassemia mutations in the (hu)beta-globin locus.
authors.affiliation: Cell and Gene Therapy Research Group, The Murdoch Childrens Research Institute, The University of Melbourne, Royal Children's Hospital, Flemington Road, Parkville, 3052 Melbourne, Australia.
authors.forename: Jim
authors.initials: J
authors.lastname: Vadolas
authors.forename: Hady
authors.initials: H
authors.lastname: Wardan
authors.forename: Marco
authors.initials: M
authors.lastname: Bosmans
authors.forename: Faten
authors.initials: F
authors.lastname: Zaibak
authors.forename: Duangporn
authors.initials: D
authors.lastname: Jamsai
authors.forename: Lucille
authors.initials: L
authors.lastname: Voullaire
authors.forename: Robert
authors.initials: R
authors.lastname: Williamson
authors.forename: Panos A
authors.initials: PA
authors.lastname: Ioannou
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D002851:Chromatography, High Pressure Liquid; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D017931:DNA Primers; D004195:Disease Models, Animal; D004588:Electrophoresis, Cellulose Acetate; D040461:Gene Components; D005816:Genetic Complementation Test; D005914:Globins / Q000235:genetics* / Q000378:metabolism*; D006454:Hemoglobins / Q000235:genetics; D006579:Heterozygote; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D051379:Mice / Q000235:genetics*; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008845:Microinjections; D010641:Phenotype; D019076:Transgenes / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D017931:DNA Primers; D006454:Hemoglobins; D005914:Globins