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To examine the relationship between erythrocyte membrane protein 7. 2b deficiency and the hemolytic anemia of human hereditary stomatocytosis, we created 7.2b knock-out mice by standard gene targeting approaches. Immunoblots showed that homozygous knock-out mice completely lacked erythrocyte protein 7.2b. Despite the absence of protein 7.2b, there was no hemolytic anemia and mouse red blood cells (RBCs) were normal in morphology, cell indices, hydration status, monovalent cation content, and ability to translocate lipids. The absence of the phenotype of hereditary stomatocytosis implies that protein 7.2b deficiency plays no direct role in the etiology of this disorder and casts doubt on the previously proposed role of this protein as a mediator of cation transport in RBC.

The human blood platelet fibrinogen receptor, integrin alphaIIbbeta3 (glycoprotein IIb-IIIa) is an archetypal member of the integrin family of adhesive molecules and is the only integrin encoded by genes physically linked in the genome. Because studies on the normal and abnormal expression of any gene require a thorough understanding of its organization, the initial goals of the current study were to determine the size and complete the genomic organization for the beta3 gene. We now report the isolation of the entire beta3 gene in a single P1 plasmid and for the first time have linked the first and second exons on a contiguous fragment of DNA. Using pulsed-field gel analysis, we determined the full size of the beta3 gene to be 63 kb and show a large (16.7 kb) first intron; based on this information, we propose a uniform numbering system for the beta3 exons. We have completed the 5' genomic structure and generated a long-range restriction map. The promoter and the 5' end of the first intron were found to have approximately 50% sequence identity with a region of the avian beta3 gene known to possess functional transcriptional activity. Analysis of three different homologous regions led to the identification of a sequence in the 5'-UTR of the human gene, CCGCGGGAGG, which shares 90% identity with the avian gene and which bound nuclear proteins in DNaseI and electrophoretic mobility shift assay studies. Mutating this sequence caused a 2.6-fold reduction in reporter gene activity. In these studies we have (1) determined the full length and 5' organization of the beta3 gene, (2) identified a large region of homology between the 5' regions of the avian and human genes, and (3) identified a sequence in the 5'-UTR that augments gene expression. Knowing the genomic structure of beta3 has permitted the uncovering of new mechanisms of mutagenesis causing Glanzmann thrombasthenia (Jin et al, J Clin Invest 98:1745, 1996), and our findings will be valuable for such genetic analyses as well as for studies on the transcriptional regulation of beta3 and other integrin genes.

Hemoglobin (Hb) S Antilles is a naturally occurring form of sickling human Hb but causes a more severe phenotype than Hb S. Two homozygous viable Hb S Antilles transgene insertions from Tg58Ru and Tg98Ru mice were bred into MHOAH mice that express high oxygen affinity (P50 approximately 24.5 mm Hg) rather than normal (P50 approximately 40 mm Hg) mouse Hbs. The rationale was that the high oxygen affinity MHOAH Hb, the lower oxygen affinity of Hb S Antilles than Hb S (P50 approximately 40 v 26.5 mm Hg), and the lower solubility of deoxygenated Hb S Antilles than Hb S (approximately 11 v 18 g/dL) would favor deoxygenation and polymerization of human Hb S Antilles in MHOAH mouse red blood cells (RBCs). The Tg58 x Tg98 mice produced have a high and balanced expression (approximately 50% each) of h alpha and h beta(S Antilles) globins, 25% to 35% of their RBCs are misshapen in vivo, and in vitro deoxygenation of their blood induces 30% to 50% of the RBCs to form classical looking, elongated sickle cells with pointed ends. Tg58 x Tg98 mice exhibit reticulocytosis, an elevated white blood cell count and lung and kidney pathology commonly found in sickle cell patients, which should make these mice useful for experimental studies on possible therapeutic intervention of sickle cell disease.

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.

zeta-Globin is the embryonic form of the alpha chain of hemoglobin. Transgenic mice generated with zeta-globin constructs containing the zeta-globin gene, 557 bp of 5' flanking sequence, and 2-kb of 3' flanking sequence linked to the beta-globin locus control region hypersensitive site 2 (HS2) expressed human zeta-globin only in embryonic yolk sac erythroid tissue, and not in definitive erythroid tissue in the fetal liver or in adult peripheral blood. To determine what sequences in the 5' flanking region of the zeta-globin gene might be important for developmental specificity, a series of 5' deletion constructs of the zeta-globin gene were made and used to generate transgenic mice. The 5' ends of these constructs were located 417, 207, and 128 bp 5' to the zeta-globin transcriptional start site, and HS2 was included to increase the level of erythroid-specific expression. In all lines of mice tested, human zeta-globin was expressed only in embryonic tissue, and not in fetal livers or in adult peripheral blood. Expression was independent of copy number and appeared to be dependent on the site of transgene insertion. These data suggest that the proximal 128 bp of the zeta-globin promoter is sufficient to properly regulate zeta-globin expression during development.