A Common Human β Globin Splicing Mutation Modeled in Mice

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A Common Human β Globin Splicing Mutation Modeled in Mice by Jada Lewis, Baoli Yang, Ronald Kim, Halina Sierakowska, Ryszard Kole, Oliver Smithies, and Nobuyo Maeda Blood Volume 91(6):2152-2156 March 15, 1998 ©1998 by American Society of Hematology

Replacement of the murine adult β globin genes by the human βIVS-2-654 gene. Replacement of the murine adult β globin genes by the human βIVS-2-654 gene. The socket-containing chromosome (A), the th-4 plug targeting construct (B), and the correctly targeted chromosome (C) are shown. The exons and introns of genes are represented as boxes and thick lines, respectively. The human β globin gene is cross-hatched with the position of the IVS-2-654 mutation shown with an asterisk. Promoter (P) and exons 1-9 of HPRT are marked. Upstream and downstream sequences that are identical or homologous in the targeting construct and the target chromosome are demarcated by dashed lines. βh3 is a β globin pseudogene. Recombination (indicated by Xs) occurs between the target locus (A) and the plug targeting construct (B), yielding a chromosome that contains the human β globin gene in place of the adult murine β globin genes, β major and β minor. Additionally, the neo gene is removed and a functionalHPRT gene is created by the correct targeting. TheHPRT gene and globin genes are transcribed from left to right in the figure, the neo gene is transcribed from right to left. Jada Lewis et al. Blood 1998;91:2152-2156 ©1998 by American Society of Hematology

A diagram and RT-PCR results showing aberrant splicing as seen in humans of the human βIVS-2-654 transcript in theHbbth-4/Hbb+ thalassemic mice. A diagram and RT-PCR results showing aberrant splicing as seen in humans of the human βIVS-2-654 transcript in theHbbth-4/Hbb+ thalassemic mice. (A) The diagram shows the human βIVS-2-654 gene with aberrantly spliced β globin mRNA produced from the mutant gene compared with the correctly spliced β globin mRNA that would have been produced from a wild-type gene. The thick line between nucleotides 580 and 652 shows the region of IVS-2 that is maintained in the βIVS-2-654 mRNA. RT-PCR primer i and iii are shown at the location and in the direction in which they anneal to the RNA. Primer i anneals to sequences within the second exon of human β globin, and primer iii anneals to two bases of the sequence at the end of the second exon and 29 bases of the region of the second intron that is maintained with this mutation in humans. (B) An autoradiograph of polyacrylamide gel electrophoresis of RT-PCR with primers i and iii on RNA from a heterozygous huβs/Hbb + mouse (lane 1), a heterozygous Hbb th-4 /Hbb + thalassemic mouse (lane 2), a HeLa cell line transfected with a βIVS-2-654 gene (lane 3), and a normal human (lane 4) is shown. Aberrantly spliced β globin mRNA RT-PCR product is 233-bp, as labeled. Correctly spliced β globin mRNA does not amplify with primers i and iii. Jada Lewis et al. Blood 1998;91:2152-2156 ©1998 by American Society of Hematology

A diagram and RT-PCR results showing aberrant splicing of the human βIVS-2-654 transcript in theHbbth-4/Hbb+ thalassemic mice. A diagram and RT-PCR results showing aberrant splicing of the human βIVS-2-654 transcript in theHbbth-4/Hbb+ thalassemic mice. (A) The diagram shows the human βIVS-2-654 gene with aberrantly spliced β globin mRNA produced from the mutant gene compared with the correctly spliced β globin mRNA that would have been produced from a wild-type gene. The thick line between nucleotides 580 and 652 shows the region of IVS-2 that is maintained in the βIVS-2-654 mRNA. RT-PCR primers i and ii are shown at the location and in the direction in which they anneal to the RNA. Primer i anneals to sequences within the second exon of human β globin, and primer ii anneals to sequences within the third exon of human β globin. (B) An autoradiograph of polyacrylamide gel electrophoresis of RT-PCR with primers i and ii on RNA from a heterozygous huβ s/Hbb +mouse (lane 1), a heterozygousHbb th-4 /Hbb + thalassemic mouse (lane 2), a HeLa cell line transfected with a βIVS-2-654 gene (lane 3), and a normal human (lane 4) is shown. Aberrantly spliced β globin mRNA and correctly spliced β globin mRNA RT-PCR products are 303 bp and 230 bp, as labeled. Jada Lewis et al. Blood 1998;91:2152-2156 ©1998 by American Society of Hematology

Peripheral blood smears from wild-type and heterozygousHbbth-4/Hbb + mice. Peripheral blood smears from wild-type and heterozygousHbbth-4/Hbb + mice. Blood smears stained with Wright stain from (a) a wild-type 129/Ola mouse and (b) an F1 heterozygous Hbb th-4 /Hbb + thalassemic mouse (600X). Jada Lewis et al. Blood 1998;91:2152-2156 ©1998 by American Society of Hematology