RESUMO
When transgenic mice that expressed human sickle hemoglobin were mated with mice having knockout mutations of the mouse alpha- and beta-globin genes, animals were produced that synthesized only human hemoglobin in adult red blood cells. Similar to many human patients with sickle cell disease, the mice developed a severe hemolytic anemia and extensive organ pathology. Numerous sickled erythrocytes were observed in peripheral blood. Although chronically anemic, most animals survived for 2 to 9 months and were fertile. Drug and genetic therapies can now be tested in this mouse model of sickle cell disease.
Assuntos
Anemia Falciforme , Modelos Animais de Doenças , Anemia Falciforme/sangue , Anemia Falciforme/genética , Anemia Falciforme/patologia , Animais , Cromatografia Líquida de Alta Pressão , Cruzamentos Genéticos , Eritrócitos/patologia , Globinas/genética , Hemoglobina Falciforme/genética , Hemoglobinas/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos TransgênicosRESUMO
beta zero-Thalassemia is an inherited disorder characterized by the absence of beta-globin polypeptides derived from the affected allele. The molecular basis for this deficiency is a mutation of the adult beta-globin structural gene or cis regulatory elements that control beta-globin gene expression. A mouse model of this disease would enable the testing of therapeutic regimens designed to correct the defect. Here we report a 16-kb deletion that includes both adult beta-like globin genes, beta maj and beta min, in mouse embryonic stem cells. Heterozygous animals derived from the targeted cells are severely anemic with dramatically reduced hemoglobin levels, abnormal red cell morphology, splenomegaly, and markedly increased reticulocyte counts. Homozygous animals die in utero; however, heterozygous mice are fertile and transmit the deleted allele to progeny. The anemic phenotype is completely rescued in progeny derived from mating beta zero-thalassemic animals with transgenic mice expressing high levels of human hemoglobin A. The beta zero-thalassemic mice can be used to test genetic therapies for beta zero-thalassemia and can be bred with transgenic mice expressing high levels of human hemoglobin HbS to produce an improved mouse model of sickle cell disease.
Assuntos
Deleção de Genes , Globinas/genética , Talassemia beta/genética , Animais , Southern Blotting , Quimera , Cruzamentos Genéticos , Modelos Animais de Doenças , Embrião de Mamíferos , Eritrócitos/citologia , Eritrócitos/patologia , Feminino , Fertilidade , Hemoglobinas/biossíntese , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Recombinação Genética , Células-Tronco/fisiologiaRESUMO
DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid 'CNC domain' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.
Assuntos
Eritrócitos/metabolismo , Regulação da Expressão Gênica , Globinas/genética , Fatores de Transcrição/genética , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Globinas/biossíntese , Células HeLa , Humanos , Leucemia Eritroblástica Aguda , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Fator 1 Relacionado a NF-E2 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.
