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Generation and characterization of the Anp32e-deficient mouse.

Reilly PT, Afzal S, Wakeham A, Haight J, You-Ten A, Zaugg K, Dembowy J, Young A, Mak TW - PLoS ONE (2010)

Bottom Line: Like other Anp32 family members, Anp32e (a.k.a.No defects in thymocyte apoptosis in response to various stresses, fibroblast growth, gross behaviour, physical ability, or pathogenesis were defined.These results provide evidence that significant functional redundancy exists among Anp32 family members.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore, Singapore.

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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Related in: MedlinePlus

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.
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pone-0013597-g001: 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.

Mentions: To determine whether Anp32e had a unique biological role, we generated a targeting construct that resulted in the constitutive elimination of Anp32e exons 2–5 in one clone of murine ES cells (Figure 1A). Southern blotting of fetal mouse DNA using flanking probes confirmed the deletion of exons 2–5 (Figure 1B). Mutant mice were generated by standard blastocyst injection and backcrossed for six generations into the C57BL6 background. Quantitative RT-PCR analysis of mRNA from MEFs and thymocytes from Anp32e+/+ and Anp32e−/− mice showed that no transcripts were expressed from the targeted Anp32e allele (Figure 1C). Using the same cDNA preparations to test for Anp32a and Anp32b mRNA expression, we found that these transcripts may be induced by deletion of Anp32e, particularly in thymocytes (Figure 1C). To more closely examine this potential induction, we tested whether protein levels of Anp32a and Anp32b were also increased in Anp32e−/− thymocytes. As shown in Figure 1D, there was no evident compensatory induction of either Anp32a or Anp32b protein levels in the absence of Anp32e.


Generation and characterization of the Anp32e-deficient mouse.

Reilly PT, Afzal S, Wakeham A, Haight J, You-Ten A, Zaugg K, Dembowy J, Young A, Mak TW - PLoS ONE (2010)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964292&req=5

pone-0013597-g001: 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.
Mentions: To determine whether Anp32e had a unique biological role, we generated a targeting construct that resulted in the constitutive elimination of Anp32e exons 2–5 in one clone of murine ES cells (Figure 1A). Southern blotting of fetal mouse DNA using flanking probes confirmed the deletion of exons 2–5 (Figure 1B). Mutant mice were generated by standard blastocyst injection and backcrossed for six generations into the C57BL6 background. Quantitative RT-PCR analysis of mRNA from MEFs and thymocytes from Anp32e+/+ and Anp32e−/− mice showed that no transcripts were expressed from the targeted Anp32e allele (Figure 1C). Using the same cDNA preparations to test for Anp32a and Anp32b mRNA expression, we found that these transcripts may be induced by deletion of Anp32e, particularly in thymocytes (Figure 1C). To more closely examine this potential induction, we tested whether protein levels of Anp32a and Anp32b were also increased in Anp32e−/− thymocytes. As shown in Figure 1D, there was no evident compensatory induction of either Anp32a or Anp32b protein levels in the absence of Anp32e.

Bottom Line: Like other Anp32 family members, Anp32e (a.k.a.No defects in thymocyte apoptosis in response to various stresses, fibroblast growth, gross behaviour, physical ability, or pathogenesis were defined.These results provide evidence that significant functional redundancy exists among Anp32 family members.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore, Singapore.

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.

Show MeSH
Related in: MedlinePlus