FIG. 4.

Generation of APOBEC3-deficient mice. (A) Targeting construct. The construct contains a Neo selective marker inserted into the third exon of APOBEC3 and includes a flanking diphtheria toxin-encoding cassette (DTA). Av, AvrII; E, EcoNI; Hc, HincII; N, NdeI; S, ScaI. (B) Targeted integration of the construct was assessed by Southern blot analysis hybridizing with probe LP to HincII/AvrII-digested DNA (as shown) as well as with probe RP to NdeI/ScaI digests (not shown). Note [as shown by the Southern blot comparison of the pattern of hybridization of probe LP to HincII/EcoNI-digested DNA from 129/Ola, C57BL/6 and (129/Ola × C57BL/6)F₁ mice (129xC57Bl/6) and consistent with sequence databases] that 129/Ola and C57BL/6 mice differ with respect to the sizes of the HincII/EcoNI (and also HincII/AvrII) bands that hybridize with LP, reflecting a 200- to 300-nt deletion in the 129/Ola APOBEC3 first intron, which removes an AvrII and an EcoNI site. wt, wild type; ht, heterozygous; ko, knockout. (C) Expression of APOBEC3 RNA in tissues of normal mice, as judged by Northern blot analysis. (D) Absence of APOBEC3 RNA in spleen of homozygous APOBEC3-targeted mice, as judged by Northern blot analysis. (E) Absence of APOBEC3 polypeptide in splenic extracts of homozygous APOBEC3-targeted mice as judged by Western blot analysis using a rat anti-mouse APOBEC3 antiserum.