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BALB/C-Congenic ANP32B-Deficient Mice Reveal a Modifying Locus That Determines Viability

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BALB/C-Congenic ANP32B-Deficient Mice Reveal a Modifying Locus That Determines Viability Exp. Anim. 65(1), 53–62, 2016 —Original— BALB/c-congenic ANP32B-deficient mice reveal a modifying locus that determines viability Vonny I. LEO1), Ralph M. BUNTE2), and Patrick T. REillY1) 1) Laboratory of Inflammation Biology, National Cancer Centre Singapore 2) Duke-NUS Graduate School of Medicine, Singapore Abstract: We previously found that deletion of the multifunctional factor ANP32B (a.k.a. SSP29, APRIL, PAL31, PHAPI2) resulted in a severe but strain-specific defect resulting in perinatal lethality. The difficulty in generating an adult cohort of ANP32B-deficient animals limited our ability to examine adult phenotypes, particularly cancer-related phenotypes. We bred the Anp32b-null allele into the BALB/c and FVB/N genetic background. The BALB/c, but not the FVB/N, background provided sufficient frequency of adult Anp32b-null (Anp32b−/−) animals. From these, we found no apparent oncogenic role for this protein in mammary tumorigenesis contrary to what was predicted based on human data. We also found runtism, pathologies in various organ systems, and an unusual clinical chemistry signature in the adult Anp32b−/− mice. Intriguingly, genome-wide single-nucleotide polymorphism analysis suggested that our colony retained an unlinked C57BL/6J locus at high frequency. Breeding this locus to homozygosity demonstrated that it had a strong effect on Anp32b−/− viability indicating that this locus contains a modifier gene of Anp32b with respect to development. This suggests a functionally important genetic interaction with one of a limited number of candidate genes, foremost among them being the variant histone gene H2afv. Using congenic breeding strategies, we have generated a viable ANP32B-deficient animal in a mostly pure background. We have used this animal to reliably exclude mouse ANP32B as an important oncogene in mammary tumorigenesis. Our further phenotyping strengthens the evidence that ANP32B is a widespread regulator of gene expression. These studies may also impact the choice of subsequent groups with respect to congenic breeding versus de novo zygote targeting strategies for background analyses in mouse genetics. Key words: chromatin regulators, congenic breeding, modifier locus, strain-dependent SNPs Introduction genetics community must plan to utilize strain-dependent phenotypes for maximal benefit, particularly with respect Although phenotypes in mice provide significant in- to identification of genetic interactions. Novel zygote sight into the role of particular genes in human develop- targeting strategies in purebred animals will facilitate ment and disease etiology, the genes are commonly hypothesis-driven research, but it may also limit op- examined in only one or two genetic backgrounds [10, portunities for genetic interaction discovery. Regardless, 24]. Strain-dependent phenotypes have long been known the BALB/c strain, being distantly related to the most to complicate developmental genetics [2, 5, 8, 11–13]. commonly used C57BL/6J strain [19] and the second As new gene-ablation systems provide for fast and ef- most frequently cited strain for analyses in mice [7], is ficient gene ablations in zygotes [25, 30], the mouse- an excellent candidate strain for examining genetic back- (Received 22 June 2015 / Accepted 16 August 2015 / Published online in J-STAGE 10 November 2015) Adress corresponding: P. T. Reilly, National Cancer Centre Singapore − Laboratory of Inflammation Biology, 9 Hospital Dr., Singapore 169610 Supplementary Tables: refer to J-STAGE: https://www.jstage.jst.go.jp/browse/expanim ©2016 Japanese Association for Laboratory Animal Science 54 V. LEO, R. BUNTE, AND P. REILLY ground effects on phenotypes. BALB/c and FVB/N wild-type mice (InVivos Pte Ltd., The acidic (leucine-rich) nuclear phosphoprotein fam- Singapore). All mice were provided 5% irradiated chow ily member B (ANP32B) is a member of the ANP32 and water ad libitum and housed under SPF conditions family of proteins that are composed of an amino termi- in individually ventilated cages. Genotyping was per- nal leucine-rich repeat domain and a carboxy-terminal formed on tail biopsies by PCR according to a previ- low-complexity acidic region [reviewed in 22]. The ously reported protocol [20]. Animals were maintained conservation of these factors combined with their over- in social housing except in cases where males demon- lapping biochemical activities suggest that they fulfill strated aggression toward cocaged mice. In such cases important activities, potentially with some redundancy. environmental differences were minimized by biweekly ANP32B (also known as SSP29, APRIL, PAL31, exchange of bedding between paired animals. Euthana- PHAPI2) has reported roles in apoptosome activation sia was carried out by carbon dioxide asphyxiation fol- [6], ELAVL1/HuR-assisted mRNA transport [3, 4], Cas- lowed by cervical dislocation. pase-3 inhibition [23, 27], and KLF5-mediated transcrip- For aging experiments, pairs of animals were exam- tional repression by means of chromatin remodelling ined daily for signs of morbidity including signs of body [15]. ANP32B is expressed in proliferating tissue [1, 26] weight loss; inability to eat or drink; behavioral abnor- and its mRNA expression is also a negative prognostic malities such as hunched posture, shivering, decreased indicator for human breast cancer [20] although ANP32B activity, or immobility; or clinical symptomatology such may also function as a tumor-suppressor protein based as ruffled fur-coat, lameness, paralysis, dyspnea, edema, on genome-wide mutation mapping [29]. not eating or drinking, and abnormal discharge. Evidence In contrast to ANP32A and ANP32E, which do not of any of these was indicative of humane endpoint. demonstrate apparent knockout phenotypes [18, 21, 30], we previously showed that ANP32B is important for Chemical cancer induction normal mouse development [20]. The Anp32b-null (An- Female experimental pairs were provided the polycy- p32b−/−) phenotype was strain dependent with almost clic hydrocarbon 7,12-dimethylbenz (a) anthracene fully penetrant lethality in the C57BL/6J congenic back- (DMBA) per oral as described in [9] with small modi- ground and semi-penetrant perinatal lethality in the mixed fications. To facilitate orogastric gavage, mice were C57BL/6J-129P2/OlaHsd background, hereafter referred maintained without chow for 30 min prior to gavage and to as the “mixed background”. In the mixed background DMBA was dosed using 0.05 ml of 20 mg/ml DMBA in the ANP32B deficiency was characterized by runtism, tricaprylin (Tokyo Chemical Industry Co., Ltd.). Animals premature aging, various pathologies and anatomical were examined daily for signs of morbidity including defects including eustachian tube defects [20]. signs of body weight loss; inability to eat or drink, be- Here, we describe the phenotyping of the ANP32B havioural abnormalities such as hunched posture, shiver- deficiency in the FVB/N and BALB/c strains. We show ing, decreased activity, or immobility; or clinical symp- that, as was seen in the C57BL/6J background, FVB/N- tomatology such as ruffled fur coat, lameness, paralysis, congenic Anp32b−/− mice occur very infrequently. In dyspnea, edema, not eating or drinking, and abnormal contrast, a large percentage of six-generation BALB/c- discharge. Evidence of any of these or evidence of a congenic ANP32B-deficient mice were viable. In addi- tumor of 2 cm in diameter was indicative of humane tion to presenting cancer and developmental data on this endpoint. background, we present the discovery a modifier locus of the Anp32b−/− viability phenotype in the BALB/c Clinical chemistry background and suggest that the modifier gene may be After CO2 asphyxiation, blood was collected by car- the variant histone gene H2afv. diac puncture from paired animals in K2-EDTA tubes (cat # 365973, Becton, Dickenson and Co., Franklin Methods and Materials Lakes, NJ, USA). Plasma was separated from leukocytes by centrifugation (4°C, 4,250 × g), snap frozen in liquid Animals nitrogen, and stored at −20°C. Samples were shipped Mice containing the Anp32b-null allele in the con- frozen for expanded toxicity analysis (cat # 60514, genic C57BL/6J background [20] were bred with IDEXX BioResearch, Columbia, MO, USA). VIABLE ANP32B DEFICIENCY REVEALS MODIFIER LOCUS 55 Immunohistochemistry Table 1. Strain-dependent survival of Anp32b-deficient mice. Tumors from DMBA-treated mice were stained with Mendelian ratios of Anp32b heterozygote intercrosses from six-generation (A) FVB/N and (B) BALB/c con- anti-cytokeratin 5 (Covance) and anti-cytokeratin 8. The genic backgrounds TROMA-1 antibody, against cytokeratin 8, developed (A) FVB/N Congenic (B) BALB/c Congenic by Rolf Kemler and Philip Brulet was obtained from the Anp32b genotype Anp32b genotype Developmental Studies Hybridoma Bank, created by the Eunice Kennedy Shriver National Institute of Child +/+ +/– –/– +/+ +/– –/– Health and Human Development (NICHD) of the Na- Expected 62 123 62 83 167 83 Observed 92 148 6 102 181 50 tional Institutes of Health (NIH) and maintained at the -15 -4 University of Iowa. (A) Chi-square test: P < 10 , (B) Chi-square test: P < 10 . Genome analysis Four random heterozygous mice were selected from parentage, all heterozygote breedings were performed separate BALB/c-congenic breeding pairs for strain- using one male and one female per cage. Consistent with specific single-nucleotide polymorphism (SNP) analysis previous analyses, we found that viability was strain (Harlan Labs). Targeted rs6190775 genotyping was per-
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