Animals

Townes mice were bred in Dr Ofori-Acquah’s laboratory at the University of Pittsburgh, using breeding pairs from Jackson Laboratories (Stock No: 013071, Townes model, hα/hα::β^A/β^S, hα/hα::-383 γ-β^A/-1400 γ-β^S). Some Townes mice were bred in Gupta laboratory at the University of Minnesota using breeders from the Ofori-Acquah laboratory. The Townes mouse “knock-in” model of SCA used in this study was developed by replacing the mouse α globin genes with a human-globin gene (hα/hα) and by replacing the mouse beta globin genes with human A-γ- and β-S-globin genes (-1400 γ-βs/-1400 γ-βs) [23]. HbSS-Townes mice have severe hemolytic anemia due to erythrocyte sickling, reticulocytosis, splenic infarcts, kidney damage, and overall poor health [23]. HbSS-BERK do not express any mouse hemoglobin and carry copies of a transgene containing human α1, γ, δ, and β-sickle genes on a highly mixed genetic background [21]. HbSS-BERK have severe disease that simulates human sickle cell anemia including hemolysis, reticulocytosis, anemia, extensive organ damage, shortened life span and pain [4, 5, 21]. Control HbAA-BERK are littermates of HbSS-BERK and therefore have the same mixed genetic background as HbSS-BERK, but exclusively express normal human hemoglobin A (human α and β globins) and no murine globins. Similarly, Townes control mice do not express mouse hemoglobin and express the human globin gene (hα/hα), human A-γ- and human wild-type β-globin (hβ/hβ) genes [23, 25].

Mice were bred and phenotyped for sickle and normal human hemoglobin by isoelectric focusing as previously described [5]. Genotyping for the knockout and hemoglobin transgenes was done by Transnetyx (Cordova, TN). Nine groups of female mice of varying ages were studied: HbSS-Townes, 4.12 ± 0.12 months, 7.19 ± 0.12 months and 10.18 ± 0.41 months; HbAA-Townes, 3.69 ± 0.0.20 months and 8.94 ± 0.19; and HbSS-BERK, 4.42 ± 0.05 months, 6.80 ± 0.09 months and 10.80 ± 0.63 months; HbAA-BERK, 8.97 ± 0.0.18 months. Seven groups of male mice of varying ages were studied: HbSS-Townes, 5.98 ± 0.66 months and 9.99 ± 0.07 months, HbAA-Townes, 5.93 ± 0.55 months and 9.34 ± 0.37 months; HbSS-BERK, 6.39 ± 0.32 months and 10.80 ± 0.63 months; and HbAA-BERK: 11.00 ± 0.99 months.