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. 2010 Oct 26;5(10):e13597.
doi: 10.1371/journal.pone.0013597.

Generation and characterization of the Anp32e-deficient mouse

Patrick T Reilly  1 , Samia Afzal, Andrew Wakeham, Jillian Haight, Annick You-Ten, Kathrin Zaugg, Joanna Dembowy, Ashley Young, Tak W Mak
Affiliations

Generation and characterization of the Anp32e-deficient mouse

Patrick T Reilly et al. PLoS One. .

Abstract

Background: Accumulated literature suggests that the acidic nuclear phosphoprotein 32 kilodalton (Anp32) proteins control multiple cellular activities through different molecular mechanisms. Like other Anp32 family members, Anp32e (a.k.a. Cpd1, PhapIII) has been conserved throughout vertebrate evolution, suggesting that it has an important function in organismal survival.

Principal findings: Here, we demonstrate that the Anp32e gene can be deleted in mice without any apparent effect on their wellbeing. No defects in thymocyte apoptosis in response to various stresses, fibroblast growth, gross behaviour, physical ability, or pathogenesis were defined. Furthermore, combined deletion of Anp32a and Anp32e also resulted in a viable and apparently healthy mouse.

Significance: These results provide evidence that significant functional redundancy exists among Anp32 family members.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation and Validation of Anp32e-deficient mice.
A. Targeting of the Anp32e gene. The diagram shows the murine genomic Anp32e locus and the targeting construct that replaced exons 2 to 6 with a pgk-neo cassette. Regions of homology in the targeting construct are shaded. Stu, StuI sites used for Southern blotting. Hind, HindIII sites used for Southern blotting of neo. FP, flanking probe. The sizes of the diagnostic Stu1 fragments for the wild-type Anp32e allele (3.1 kb) and the targeted Anp32e allele (1.5 kb) as well as the diagnostic HindIII fragment for neo insertion (4.1 kb) are shown. B. Confirmation of deletion. DNA from fetal Anp32e+/+ (wt), Anp32e+/− (E+/−), and Anp32e−/− (E−/−) mice was subjected to Southern blotting using the flanking probe in shown in diagram 1A (within intron 6). DNA from Anp32e+/+ (wt) and Anp32e+/− (E+/−) ES cells was also subjected to southern blotting with a neo probe in order to confirm a single vector insertion. C. Validation of Anp32e mRNA deficiency. Quantitative RT-PCR of mRNA from primary MEFs from Anp32e+/+, Anp32e+/− and Anp32e−/− embryos at E14.5, and (bottom) thymocytes from Anp32e+/+, Anp32e+/− and Anp32e−/− mice at 4–8 weeks of age. Expression levels of Anp32a (black bars), Anp32b (grey bars), and Anp32e (white bars) mRNAs are shown relative to levels in Anp32e+/+ mice. Error bars represent the standard deviation from the mean across three technical replicates per sample. D. Absence of compensatory induction of other Anp32 proteins. Protein extracts from lymphocytes of Anp32e+/+ (wt) and Anp32e−/− (E−/−) mice were probed for Anp32a, Anp32b, and beta-tubulin expression.
Figure 2
Figure 2. Normal proliferation and apoptosis of Anp32e−/− cells.
A. Growth curves of primary MEFs from Anp32e+/+, Anp32e+/− and Anp32e−/− mice (n = 2/genotype). Nt/N0, cell number at time point/cell number on day 0. B. Apoptosis of Anp32e+/− and Anp32e−/− thymocytes after 20 hours in culture response to (left to right): –ve, untreated controls, UV-irradiation (30 mJ/cm2 and 60mJ/cm2), γ-irradiation (1 Gy and 2 Gy), etoposide (1 µM and 3 µM), dexamethasone (3 nM and 10 nM), and staurosporine (1 µM and 3 µM). Results shown are mean % viable cells ± SD (n = 3/genotype).
Figure 3
Figure 3. Normal rotorod behaviour.
Individual Anp32e+/+ and Anp32e−/− mice (spherical symbols) were placed on a rotating rotorod and the time they remained suspended was measured in seconds. Horizontal bars, mean duration per genotype.
Figure 4
Figure 4. Generation and Validation of Anp32a-deficient mice.
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. RV, EcoRV; X, XbaI; Spe, SpeI; FP, 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.
Figure 5
Figure 5. Growth Curve of Anp32a-deficient fibroblasts.
Growth curves of early passage primary MEFs from littermate isolates of Anp32a+/+ and Anp32a−/− embryos. Two isolates per genotype to give four littermate MEF lines total. Nt/N0, cell number at time point/cell number on day 0.
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