Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.

Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.