24 TARGETED DISRUPTION OF ATAXIA-TELANGIECTASIA MUTATED GENE IN MINIATURE PIGS BY SOMATIC CELL NUCLEAR TRANSFER

Abstract

Ataxia-telangiectasia (A-T) is a genetic disease caused by germline biallelic mutation in the ataxia-telangiectasia mutated gene (ATM) that results in partial or complete loss of ATM expression or activity. A diverse phenotype of the disease includes progressive cerebellar ataxia, oculocutaneous telangiectasias, radiation hypersensitivity, increased cancer incidence, immunodeficiency and chromosomal instability. The frequency of A-T in the United States and United Kingdom has been estimated to be 1:40 000. Heterozygous carriers may also have predisposition to diverse cancers. Although ATM-deficient mice have been produced, none reflects the extent of neurological abnormalities involving the loss of Purkinje cells of the cerebellum seen in patients. Hence, miniature pigs, which are anatomically and physiologically closer to humans, might serve as an alternative model for human A-T. In the present study, we attempted a targeted disruption of ATM in miniature pigs by somatic cell nuclear transfer. Most A-T patients possess mutated ATM with truncation, resulting in prematurely terminated ATM proteins that are highly unstable. To induce the truncation of ATM, we disrupted exon 59 region of ATM that has been known to be critical for ATM kinase activity. Miniature pig fetal fibroblasts were transfected with ATM-targeting vector and treated by neomycin for 2 weeks. A total of 139 colonies were screened by PCR and 3 among them were identified as homologous recombinants with monoallelic disruption of ATM (targeting efficiency = 2.1%). One of the 3 colonies was chosen and used for subsequent nuclear transfer. In total, 611 nuclear transfer embryos reconstructed with ATM-targeted fetal fibroblasts were transferred into 5 surrogate gilts. Two gilts became pregnant and developed to term with a total of 5 live piglets delivered. Being analysed by PCR, all piglets born were found to be ATM gene-targeted. Because conventional murine models of human A-T have provided only limited insights into therapies and pharmacological treatments, the generation of miniature pigs with disrupted ATM will allow new opportunities to more precisely understand A-T and to accelerate discovery of strategies for prevention and treatment of the disease. In addition, such animals may be utilised in studying cancer with respect to the involvement of ATM in cell cycle arrest, DNA repair and apoptosis.