Generation of CD38 gene-targeted mice. (A) Schematic diagram of the CD38 genomic locus, targeting vector, and targeted locus. Exons, lengths of regions of CD38 homology, lengths of diagnostic restriction fragments, and the position of the exon 4-specific probe are indicated. Restriction enzyme sites are indicated (H, HindIII; C, Cla I; S, Sac I; S*,Sac I polymorphism between the 129SvEv and BALB/c strains of mice). Arrows indicate the transcriptional orientation of the CD38, HSV-TK, and neomycin resistance genes. Homologous recombination resulted in replacement of a 1.6-kb CD38 genomic region that included the putative NAD⁺ glycohydrolase enzyme catalytic site. (B) Southern blot analysis used for screening genomic DNA from recombinant clones (not shown), and 129Ola × C57BL/6J CD38^+/+, CD38^+/−, and CD38^−/− mice. Probing of blots with a 174-bp polymerase chain reaction–derived exon-4–specific probe (top panel) shows the predicted 2-kb and 10-kb Sac I fragments diagnostic of the CD38 endogenous and mutant alleles, respectively. As a consequence of theSac I polymorphism in the BALB/c locus, a Sac I digest of BALB/c genomic DNA provides a control for the approximate size of the mutant allele fragment. Probing of blots with a neo probe (lower panel) shows hybridization specifically to the 10-kb Sac I fragment characteristic of the mutant allele. (C) FACS staining of total splenocytes with the phycoerythrin (PE)-conjugated rat antimouse CD38 monoclonal antibody NIMR5 (0.5 μg/mL, shaded), or an appropriate PE-conjugated rat IgG2a isotype control (unshaded), was performed according to standard methods.