Complementation of α-thalassaemia in α-globin knockout mice with a 191 kb transgene containing the human α-globin locus

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Abstract

α-Thalassaemia is an inherited blood disorder caused by a decrease in the synthesis of α-globin due to mutations in one or both of the α-globin genes located on human chromosome 16. A 191 kb transgene derived from a sequenced bacterial artificial chromosome (BAC) clone carrying the human α-globin gene cluster, together with about 100 kb of sequence upstream of DNase1 hypersensitive site HS-40 and 30 kb downstream of the α1-globin gene, was introduced into fertilised mouse oocytes by pronuclear microinjection. Three transgenic founder mice were obtained. Analysis of one transmitting line by fluorescent in situ hybridisation and quantitative PCR demonstrated a single copy integration of the human α-globin transgene on chromosome 1. Analysis of haemoglobins from the peripheral blood by cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) demonstrated synthesis of human α-globin to about 36% of the level of each mouse α-globin locus. Breeding of transgenic mice with mice heterozygous for a knockout (KO) deletion of both murine α-globin genes showed that the human α-globin locus restored haemoglobin levels and red cell distribution width to normal in double heterozygous mice and significantly normalised other haematological parameters. Interestingly the human transgene also induced a significant increase in red cell production and haematocrit above wild type values. This is the first report demonstrating complementation of a murine α-globin KO mutation by human α-globin gene expression from an intact human α-globin locus. The transgenic mouse model described in this report should be very useful for the study of human α-globin gene regulation and for the development of strategies to down regulate α-globin production as a means of ameliorating the severity of β-thalassaemia.

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