Testing recombinant APOBEC2 preparations for deaminase activity. (A) His-tagged APOBEC2 and MBP-APOBEC2 fusion proteins were purified from E. coli extracts by affinity chromatography, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, stained with Coomassie blue, and Western blotted with a rabbit anti-APOBEC2 antiserum. Numbers indicate approximate sizes in kilodaltons. (B) No detectable activity of recombinant APOBEC2 on dC in DNA, as judged by the use of a variety of single-stranded 5′-labeled oligodeoxyribonucleotide substrates (317 to 330 as in reference 2) containing two clusters of dC residues (cluster A and cluster B) in different sequence contexts. Following successful deamination (as obtained using the recombinant MBP-APOBEC1 control), the labeled oligonucleotide will yield uracil-containing derivatives which, following uracil-DNA glycosylase/endonuclease treatment and purification on streptavidin-agarose, give rise to shorter, labeled oligonucleotide products. (C) The deoxycytidine deaminase activity present in recombinantAPOBEC2 samples is inhibitable by THU and is undetectable in recombinant APOBEC2 samples prepared from a cdd dcd E. coli host (results for wild-type [wt] E. coli are also shown). Deoxycytidine activity was monitored in recombinant APOBEC2 samples (2 μg) and is expressed as the percentages of the radioactivity in the [(dU)/(dU+dC)] spots following thin-layer chromatography.

Analysis of APOBEC2-deficient mice. (A) Breeding of APOBEC2-targeted mice generates homozygous APOBEC2-deficient mice which are fertile. The data are a compilation of breedings, with the litters deriving from five distinct sets of breedings. (B) Histological analysis of calf and heart muscles reveals no difference between control and APOBEC2-deficient mice. Magnifications are indicated.

(A) Breeding of APOBEC3-targeted mice generates homozygous APOBEC3-deficient mice which are fertile. The data are a compilation of breedings established with mice produced from two independent targeted ES cell clones, with the litters deriving from eight distinct sets of breedings. (B) The distribution of splenic lym-phoid subsets is unaffected by APOBEC3 deficiency. The relative proportion of T and B lymphocytes was analyzed by staining with anti-CD3 and anti-CD45R(B220), with T- and B-cell subpopulations further analyzed using antibodies to CD4/CD8 and immunoglobulin M (IgM). Myeloid cells were analyzed by staining for CD11b and Gr-1.

Generation of APOBEC2-deficient mice. (A) Targeting construct. The construct contains a Neo selective marker inserted into the second exon of APOBEC2 and includes a flanking diphtheria toxin A-encoding cassette (DTA) to allow selection for homologous integration. A, AatII; K, KpnI; N, NdeI; Sx, SexAI; Sp, SpeI; X, XbaI. (B) Targeted integration of the construct was assessed by Southern blot analysis hybridizing with probe RP to KpnI/SexAI-digested DNA (as shown) as well as with probe LP to NdeI/XbaI digests (not shown). wt, wild type; ht, heterozygous; ko, knockout. Numbers indicate approximate size in kilobases. (C and D) Expression of APOBEC2 RNA in tissues of normal mice and humans as judged by Northern blot analysis. (E) Absence of APOBEC2 RNA in heart and skeletal muscle of homozygous APOBEC2-targeted mice as judged by Northern blot analysis. (F) Absence of APOBEC2 protein in heart and skeletal muscle of homozygous APOBEC2-targeted mice as judged by Western blot analysis of whole-tissue extracts using a rabbit anti-APOBEC2 antiserum. Numbers indicate aproximate sizes in kilodaltons.

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.