Disorders of human globin gene expression are among the most common hereditary diseases of man. Detailed analysis of specific mutations in the Beta globin gene cluster has provided insights into the mechanisms of human gene regulation, and may assist in the development of genetically-based therapies. Gene transfer studies in cell lines and transgenic mice have identified important promoter and enhancer elements of the Beta globin locus. However, it has not been possible to study the physiological role of these cos-acting sequences within the intact Beta globin gene region, due to the insert size limitations of existing plasmid and cosmid vectors. The Beta globin locus, including the 5' and 3' erythroid-specific Dnase I hypersensitive sites, spans at least 90kb, whereas cosmid vectors accommodate less than 50 kb. Our goal is to more precisely define the cis-acting sequences involved in the tissue- and stage-specific expression of the members of the Beta globin gene family. The recently developed technique for cloning genomic DNA fragments of several hundred kb or more into yeast artificial chromosomes (YACs), has now made it possible to isolate large genetic loci, such as the Beta globin gene cluster, including cis-acting sequences located in 5' and 3' flanking regions. We will combine the advantages of YAC technology with gene transfer methods, in order to introduce YACs containing the Beta globin gene locus into murine cell lines and transgenic mice. Previously, we have extensively characterized two YACs containing the entire Beta globin locus and flanking regions in a single contiguous insert. Using these YACs, we will adapt methods for reliably introducing high molecular weight DNA into cells, while preserving the genomic structure of these fragments after their integration into the host chromatin. We will assess the relative advantages of different transfection procedures using murine erythroleukemia cells or embyonic stem cells. We will also test microinjection of oocytes for this purpose. Once we have produced cell lines or mice carrying the entire human Beta globlin gene locus, we will use quantitative RNA analysis to study the expression of the intact human Beta globin gene family. We will use homology-directed recombination in yeast to generate mutations of potential regulatory sequences in, and flanking the locus. Yacs containing Beta globin loci with specific mutations will be transferred using the procedures we have developed. The effect of these mutations on tissue- and developmental-stage specific gene expression will then be analyzed. We believe this work will provide a model system for better understanding the molecular mechanisms of gene regulation during ontogeny, as well as for studying structure/function relationships of large genetic loci, whose size has up to now precluded mutational analysis and transfer into cells.
