Establishment of Pen KO mice and comparison of RBC numbers. (A) Creation of a KO allele by homologous recombination. The exon/intron structure of mPen is shown at the top. The targeting vector employs a self-excising Cre-Neo cassette in which the expression of Cre is under the control of a testis-specific promoter. The locations of the genotyping primers are also shown. (B) Genotyping of the KO littermates. GT1/GT3 amplify the KO but not the WT allele due the long distance between the primer sites in the WT allele. (C) A scatter plot of RBC numbers of young mice matched in age (3 months) and sex. No significant difference is noted between Pen^+/+ and Pen^−/− mice at this age. Bars indicate means. (D) A scatter plot of RBC numbers of older mice matched in age (6 to 17 months) and sex. Significant difference exists between Pen^+/+ (○) and randomly selected Pen^−/− (▴) groups (P = .004 by paired t test). Mice with increased (more than 4%) basophilic macrocytes (▵) in prescreening were plotted separately. Significant difference is again noted between Pen^+/+ (○) and the prescreened Pen^−/− mice (▵) (P < .001 by unpaired t test). Pen^−/− mice with very severe anemia (hematocrit less than 0.20 [20%]) are not included in this analysis. Bars indicate means.