The Expression Of YAC-Mediated Human β-Globin Gene Cluster With HS2-Specific Mutations In Transgenic Mice

The eight functional globin genes and three globin pseudogenes are arranged in two gene clusters: α-and β-globin gene cluster. The α-globin gene cluster is locates on the short arm of chromosome 16 and arranged in the order of 5'- ζ -ψζ -ψα-α2-α1-θ-3', spanning 40kb; the β-globin gene cluster is on the short arm of chromosome 11 and in the order of 5'-ε-Gγ-Aγ-ψβ-δ-β-3',spanning about 75 kb. The DNA sequences of the two clusters have been determined.The expression of globin genes are characterized by 1) sequential expression of individual gene in each cluster, 2)erythroid-specific and developmental stage-specific expression, 3)balance between α-like and β-like globin chains. The transcription of globin genes is regulated by the chromatin structure, methylation of globin genes and their flanking regions, local and distal cis-acting elements and cellular trans-acting factors.The β-globin gene locus control region (LCR) consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4)which are necessary for the high level expression of globin genes in erythroid tissues. The single 5'HS2, when linked to β-globin gene, can act as an erythroid enhancer in transfected cells and transgenic mice.Studies of expression regulation of eukaryotic genes in transgenic mice permit not only the devices of experiments on molecular level but also the observation of the involvement of almost all the factors affecting gene expression and investigations of in vivo effect of gene regulations. Transgenic technique can be applied to set up transgenic models for human genetic diseases from which expression regulation of abnormal genes are demonstrated and many pre-clinical data for human gene therapy will be provided. Moreover, yeast artificial chromosome (YAC) cloning and site-specific mutation techniques can be combined with transgenic technique to investigate the developmental gene regulation of large genes (clusters) with mutations in different sites.To study the effect of LCR 5'HS2 mutation on the developmental expression of human β-globin genes, PRS306/HS2 target plasmid was designed and constructed. A normal 1.85kb HS2 fragment was isolated from plasmid pBlue-HS2 by restriction endonuclease, then linked with blunt-ended...