(YACs) in studies of mammalian development: Production of f3-globin locus YAC mice carrying human globin developmental mutants

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of ,B-globin locusYAC developmental mutants: (i) mice carrying a G -> A transition at position -117 of the Ay gene, which is responsible for the Greek Ay form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) 18-globin locus YAC transgenic lines carrying 8and ,B-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and 6j3-thalassemia syndromes. The mice carrying the -117 Ay G -> A mutation displayed a delayed yto j3-globin gene switch and continued to express Ay-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the y-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development. Molecular biological techniques have allowed the cloning and characterization of several mammalian developmental genes, and transgenic mice have been used extensively in the analysis of regulatory mechanisms controlling these genes. Although a powerful technique, gene transfer of recombinant constructs to generate transgenic mice suffers from several limitations. The size of the DNA molecule that can be injected has been constrained by limitations on the size that could be cloned and purified intact. Thus, presumably nonessential sequences are omitted in the design of constructs to be injected, and decisions are made about the regulatory relevance of the omitted sequences and the distances between cis control elements. To overcome these limitations, we have generated transgenic mice using purified yeast artificial chromosomes (YACs) (1). YACs offer two major advantages for the analysis of gene regulation. The first advantage is the insert size that can be contained within the YAC; the 100-kb to Mb size range allows the study of complete genes or multigene loci in the context of their native sequence environment. The second advantage is the ability to introduce site-directed mutations into sequences inserted in the YAC vector using the homologous recombination system of yeast harboring the YAC. We utilized a YAC containing the multigenic human 13-globin locus for studies of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. developmental regulation of gene expression in transgenic mice (1). Our data show that the genes of the 13-globin locus YAC (,B-YAC) are correctly regulated during development in the mouse (1), thus demonstrating the usefulness of the YAC/transgenic mouse system. In this work, we test whether YACs can be used for the analysis of developmental regulation by introducing mutations into the ,3-globin locus that affect cis-acting sequences known to cause aberrant developmental phenotypes. We demonstrate the usefulness of this system by introducing a point mutation that recreates the phenotype of the Greek Ay form of hereditary persistence of fetal hemoglobin (HPFH) and by analyzing the effects of deletions of the 3' end of the 3-globin locus. Our results indicate that mutant YACs can be used in the analysis of the cis control of developmentally regulated genes. MATERIALS AND METHODS Production of the -117 Aym YAC. A 5.4-kb Ssp I-Sal I fragment containing the -117 A.n gene was isolated from pUC19 A-ym 117(+) (K.R.P., unpublished results) and ligated into Sma I-Sal I-digested and phosphatase-treated yeast integrating plasmid (YIP) vector pRS406 (Stratagene) to produce pRS406 -117 A jm. Ten micrograms of pRS406 -117 Aym was linearized at a unique HindIII site in the Ay insert sequence and transformed into spheroplasted Saccharomyces cerevisiae strain AB1380 containing the YAC yneo,3globin (,B-YAC; ref. 2). Transformants were selected for uracil prototrophy on complete minimal medium lacking uracil. YIP vector sequences and one copy of the duplicated Ay gene region were excised as follows. Yeast isolates were grown in complete minimal broth lacking tryptophan and lysine (to keep selection on the YAC arms) but containing uracil (to allow spontaneous excisions of the YIP vector via homologous recombination between the duplicated Ay gene regions). Yeast cells (106_107) were plated on 5-fluoroorotic acid plates to select for spontaneous YIP excision events (frequency of 10-4-10-5). Single colony isolates were further purified and retested for loss of the URA3 gene and 5-fluoroorotic acid resistance. Purification of the -117 A_yn YAC. -117 A_/, YAC DNA was purified essentially as described (1, 2) with the following modifications. Agarose plugs were not agitated during preparation to reduce shear of DNA molecules contained within the blocks (3). The entire 3to 3.5-g gel slice was treated with agarase overnight. The supernatant (up to 4 ml) was passed through a molecular weight cut-off filter (Millipore; 100,000 nominal molecular weight limit) to concentrate the DNA Abbreviations: YAC, yeast artificial chromosome; HPFH, hereditary persistence of fetal hemoglobin; LCR, locus control region; HS, hypersensitive site; ,3-YAC, 13-globin locus YAC; YIP, yeast integrating plasmid. tTo whom reprint requests should be addressed.