Generation and Validation of Anp32a-deficient mice.

<p>A. Targeting of the Anp32a gene. The diagram shows the murine genomic Anp32a locus and the targeting construct that replaced exons 2 to 5 with a pgk-neo cassette. Regions of homology in the targeting construct are shaded. <i>RV,</i> EcoRV; X, XbaI; Spe, SpeI; <i>FP</i>, flanking probe used for Southern blot analysis. B. Confirmation of deletion. DNA from embryos of Anp32a^+/+ (wt), Anp32a^+/− (A^+/−) and Anp32a^−/− (A^−/−) was subjected to Southern blotting using the flanking probe in A (within intron 1). Anp32a^+/+(wt) and Anp32a^+/− (A^+/−) ES-cell DNA was also probed for the neomycin resistance gene (Neo probe). C. Validation of Anp32a protein deficiency by immunoblotting of extracts from mouse liver and spleen. Primary anti-APRIL antibody raised in mice against recombinant human Anp32b protein recognizes both the murine Anp32b protein and the Anp32a protein, which can be distinguished by size. Tubulin, loading control. *, non-specific bands seen in liver and spleen.