Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb β ^s -globin yeast artificial chromosome: A mouse model of sickle cell anemia

Abstract

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine ␣-and ␤-globin genes and substitution with human ␣ and ␤ s transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human ␣-, ␥-, and ␤-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human ␤-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the ␥-and ␤-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb ␤ The biochemical basis for sickle cell anemia (SCA) was described more than 30 years ago (1, 2); however, advances in the treatment of SCA have in part been hampered by the lack of an animal model that accurately reproduces the pathophysiology and genetics of this disorder. The strategies for making a murine model of SCA have evolved as the limitations of each approach became apparent. Thus, initial efforts focused on the transfer of human ␤ s -globin genes to generate transgenic lines. Heterotetramers of murine ␣-globin and human ␤ s -globin do not polymerize efficiently; even with the addition of human ␣-globin transgenes only a small fraction of the cells sickled in vivo because of the disruption of HbS by murine ␣-and ␤-globins (3-11). Under hypoxic conditions there is more extensive deoxygenation of murine hemoglobin than HbS, as mouse hemoglobin has a lower O 2 affinity than does HbS (12). To produce a hemoglobin that would polymerize more readily, two additional mutations were introduced into ␤ s transgenes; a second mutation in codon 23 to reproduce the ␤ s Antilles allele, and a third mutation to yield ␤ s AntillesD Punjab or HbSAD (6,7,13). While the SAD mice exhibited a greater propensity for red cell sickling under hypoxic conditions, this model did not fully recapitulate the features of sickle cell disease. Hence, recent efforts have been directed toward reducing or eliminating endogenous mouse globin gene expression and the production of mice that exclusively express human HbS as adults.Two such models have been reported recently (15, 16). Expression of the adult murine ␣-and ␤-globin genes was eliminated by targeted disruption of these loci in embryonic stem (ES) cells. Mice exclusively expressing human HbS were identified after successive cycles of crossbreeding knockout lines wi...

Multiple interactions between regulatory regions are required to stabilize an active chromatin hub

“…However, it is present in the globin CH at all stages analyzed to date. In addition, it is also known that the upstream HS-111 (the equivalent of mouse HS-62.5/HS-60, Bulger et al 2003; also not present in all of the lines reported here) and downstream 3Ј HS1 are not required for a high level of expression of the ␤-globin locus when introduced in transgenic mice (Strouboulis et al 1992) and that such lines express at the same level as lines that contain the 5Ј and 3Ј flanking sites (Bungert et al 1995;Peterson et al 1996;Chang et al 1998;Calzolari et al 1999). At first sight, these data would suggest that the HSs involved in the CH complex formation are not very important, but why then would there be a CH?…”

Correction of the sickle cell mutation in embryonic stem cells

“…The sickle cell anemia mouse line carrying a yeast artificial chromosome containing 240 kb of human ␤-globin gene cluster was used in these experiments (13). Female mice carrying homozygous or heterozygous mouse ␣-globin, heterozygous mouse ␤-globin gene knockouts, and homozygous human ␣-and ␤ S -globin yeast artificial chromosome transgenes were mated with male mice with the same genotype.…”

“…13. Briefly, 20 g of construct was linearized at the NotI site and electroporated into 3 ϫ 10 6 ES cells in 0.8 ml of Hepes-buffered saline in a 0.4-cm gap cuvette with a single pulse of 240 V and 125 F in a Gene Pulser (Bio-Rad).…”

“…There are several mouse models of sickle cell anemia, all carrying the human ␣-, ␤ S -, and ␥-globin transgenes and knockouts of the endogenous mouse ␣-and ␤-globin genes (11)(12)(13). Although some of the models were made by injecting truncated ␤-globin gene complex under the control of the locus control region (LCR), the one that we have made carries a ␤ S -globin transgene within a 240-kb yeast artificial chromosome that contains the LCR and the -, G ␥-, A ␥-, ␦-, and ␤ S -globin genes in their native context.…”

Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting

“…20 Briefly, concentrated chromosomal DNA from the b S -YAC strain (AB1380 background) 21 was prepared and resolved on a 1% low-melt agarose pulsed-field gel at 200 V, 141C, 20-50 s, 33 h. The YAC was isolated, equilibrated with a modified agarase buffer (10 mM BisTris-HCl pH 6.5, 1 mM EDTA, 100 mM NaCl), treated with b-agarase I (New England Biolabs), and concentrated to a final volume of B200 ml. A volume of 30 ml of the purified YAC were introduced into competent LSY678IntHyg(rep) cells by spheroplast transformation and selection on agar/sorbitol plates lacking tryptophan.…”