Targeted disruption of Ataxia-telangiectasia mutated gene in miniature pigs by somatic cell nuclear transfer

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Highlights

Abstract

Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None of the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.

Introduction

Ataxia telangiectasia (A-T) is a recessive autosomal disorder in humans with an average frequency of 1:40,000–1:100,000 live births worldwide [1], [2], [3]. It is characterized by a wide variety of pleiotropic phenotypes, including progressive cerebellar degeneration, gonadal atrophy, growth retardation, lymphoreticular malignancies, and incomplete sexual maturation. Patients with A-T are extremely sensitive to ionizing radiation, which causes double strand breaks (DSB) in the DNA [4], [5]. One of the major clinical symptoms of A-T is cerebellar ataxia, which results from the gradual loss of the Purkinje cells in the cerebellum [6], [7]. The disease is progressive, and death generally occurs by the second or third decade of life due to neurologic deterioration or lymphoreticular malignancies, which occur in 10–15% of patients [8]. Ataxia telangiectasia is caused by a deficiency or malfunction of the ataxia telangiectasia mutated (ATM) protein that normally mediates cellular response to DNA damage through multiple transduction pathways. Carriers of the ATM mutation have been estimated to comprise 0.5–1% of the general population [9], and these individuals show none of the severe clinical symptoms seen in A-T patients but do have a predisposition to cancer, particularly breast cancer in women [10].

The ATM gene has 65 exons extending over 160 kb of genomic DNA. It produces a 13-kb transcript that encodes a 350-kDa protein containing highly conserved catalytic domains for phosphatidylinositol-3-kinase, which is presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage [11], [12]. This protein initiates a signaling cascade through the phosphorylation of various downstream genes such as p53, Chk2, Mdm2, BRCA1, H2AX, and Pin2/TRF1, that in turn control cell cycle-check point, DNA double-strand break repair pathways, apoptosis, and telomere metabolism [13], [14], [15].

A murine model for A-T has generally been used to characterize the effects of the ATM mutation. Many types of ATM knockout (KO) mice have been generated to study the function of the ATM protein as well as the molecular basis of this pleiotropic and multisystemic disease [16], [17], [18]. ATM-deficient mice display a variety of symptoms similar to those of human A-T patients, including growth and meiotic defects, immunological abnormalities, radiation hypersensitivity, and predisposition to cancer, which together are the most common pleiotropic roles of ATM. However, the neurological defects in these mice develop more slowly than those clinically observed in humans, and the mice have not died from ataxia, but from other consequences of ATM-deficiency, such as lymphoma.

The neurological abnormalities seen in ATM-mutant mice do not present the same severity as human neurological abnormalities, suggesting that an alternative animal model for human A-T could be beneficial [19]. As such, the pig has already been recognized to be a useful, well-established model for medical studies of human disease. Since the pig has been able to provide insight into the mechanism and treatment of various diseases (such as cancer, diabetes, and atherosclerosis), the ATM gene-targeted pigs could be a valuable model for studying A-T. Moreover, the structure and transcription of the porcine ATM gene has already been characterized [19]. Genomic sequencing of the porcine ATM gene revealed that a similarity between humans and pigs was greater than that between humans and mice, suggesting that, from a molecular standpoint, a porcine model may be more suitable.

The aim of the present study is to produce a novel disease model for ATM that can replace the mouse model that does not fully represent all phenotypes of the disease. We therefore report on the production of cloned miniature pigs via somatic cell nuclear transfer (SCNT) using ATM gene-targeted fetal fibroblasts.

Section snippets

Animal ethics

All procedures in this study were carried out in accordance with the Code of Practice for the Care and Use of Animals for Scientific Purposes, as approved by the Institutional Animal Care and Use Committee of Dankook University.

Preparation of porcine fetal fibroblasts

Pig fetuses at 28–39 days of gestation were obtained from Minnesota miniature pigs maintained in specific pathogen-free (SPF) conditions at Seoul National University. The head, dorsal spine of the medial section, and tail of the fetuses were removed. Then, small pieces of

Generation of ATM gene-targeted fibroblasts

To disrupt the porcine ATM gene, both male and female fetal fibroblasts were separately transfected with a conventional targeting vector (Fig. 1). After antibiotic selection, 87 male and 138 female G418-resistant colonies were obtained. Among these colonies, one male and three female fibroblast colonies were identified as homologous recombinants based on PCR analysis. As shown in Fig. 2, one female colony containing normal karyotype (2n = 38, XX) was chosen for subsequent SCNT experiment.

Production of ATM gene-targeted pigs

A total

Discussion

In the present study, we described a novel large animal model for preclinical research of A-T disease. We produced heterozygous ATM knockout miniature pigs via SCNT using ATM gene-targeted cells as nuclear donors. The ATM mutations were broadly categorized to be of two types: the missense mutation (ATMmis) and the truncating mutation (ATMtrunc). The former includes non-silent mutations that cause a substitution or short in-frame insertion or deletion of amino acids. The ATM protein is expressed

Acknowledgments

This work was supported by the Korea Health Technology R&D Project (grant number HI13C0954) through the Korea Health Industry Development Institute, funded by the Korea Ministry of Health and Welfare; the Bio & Medical Technology Development Program (grant number 2014034046); and the Priority Research Centers Program (grant number 2009-0093829) through the National Research Foundation (NRF) funded by the Korea Ministry of Science, ICT and Future Planning.

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  • Cited by (6)

    1

    Present address: Institute of Green Bioscience and Technology, Seoul National University, Pyeongchang 232-916, Republic of Korea.

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