Skip to main content
letter
. 2016 Oct;101(10):e397–e400. doi: 10.3324/haematol.2016.148106

Figure 1.

Figure 1.

Generation of humanized calpain-1 knockout Townes sickle mice. (A) Schematic representation of the breeding strategy used to develop calpain-1 knockout Townes sickle (SSCKO) mice. (B) PCR/Gel electrophoresis confirming the genotypes of AA, SS, and SSCKO mice (a: human α-globin (330bp), b: human β-globin (320bp), c: absence of mouse β-globin (432bp), d: calpain-1 (650bp), e: absence of PGK-Neo cassette inserted to knockout calpain-1 (415bp), f: human α-globin (330bp), g: human sickle βs-globin (250bp), h: absence of mouse β-globin (432bp), i: calpain-1 (650bp), j: absence of PGK-Neo cassette inserted to knockout calpain-1 (415bp), k: human α-globin (330bp), l: human sickle βs-globin (250bp), m: absence of mouse β-globin (432bp), n: absence of calpain-1 (650bp), o: presence of PGK-Neo cassette inserted to knockout calpain-1 (415bp). For each mouse, PCR genotyping was conducted on 3 loci: α-globin, β-globin, and calpain-1, and the results are represented as genotypes in parentheses. For example, the CKO mouse has two alleles for the mouse alpha globin gene (+/+), two alleles for the mouse beta globin gene (+/+), and genetic inactivation of calpain-1 (−/−). The humanized Townes SS mouse has two alleles for the human alpha globin gene (α/α), two alleles for the human sickle hemoglobin gene (βS/βS), and intact calpain-1 gene (+/+). Blue: male; Pink: female. Calpain-1 is only the second gene to be systematically deleted successfully in the Townes sickle mice. (C) SS and SSCKO mice after 15 generations. (D) Phenotypic analysis of hemoglobins in different groups of Townes mice. Differential hemoglobin electrophoresis on cellulose acetate membrane was performed on each mouse used in this study. The Ponceau S stained bands show the presence of human sickle hemoglobin only in homozygous sickle mice (labeled SS and SSCKO), normal human hemoglobin A only in control mice (labeled AA) and both human sickle and normal human hemoglobin A (labeled AS) in heterozygous mice. Representative images from several mice phenotyped are shown. (E) Casein zymography confirming the absence of calpain-1 enzymatic activity in the RBCs/reticulocytes of SSCKO mice compared to their AA and SS counterparts.