Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1(-/-) and SURF1(-/+) pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1(-/-) pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1(-/+) and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1(-/-) tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down regulated in SURF1(-/-) skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1(-/-) piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.

SURF1 knockout cloned pigs: Early onset of a severe lethal phenotype

Bottani, E.;
2018

Abstract

Leigh syndrome (LS) associated with cytochrome c oxidase (COX) deficiency is an early onset, fatal mitochondrial encephalopathy, leading to multiple neurological failure and eventually death, usually in the first decade of life. Mutations in SURF1, a nuclear gene encoding a mitochondrial protein involved in COX assembly, are among the most common causes of LS. LSSURF1 patients display severe, isolated COX deficiency in all tissues, including cultured fibroblasts and skeletal muscle. Recombinant, constitutive SURF-/- mice show diffuse COX deficiency, but fail to recapitulate the severity of the human clinical phenotype. Pigs are an attractive alternative model for human diseases, because of their size, as well as metabolic, physiological and genetic similarity to humans. Here, we determined the complete sequence of the swine SURF1 gene, disrupted it in pig primary fibroblast cell lines using both TALENs and CRISPR/Cas9 genome editing systems, before finally generating SURF1(-/-) and SURF1(-/+) pigs by Somatic Cell Nuclear Transfer (SCNT). SURF1(-/-) pigs were characterized by failure to thrive, muscle weakness and highly reduced life span with elevated perinatal mortality, compared to heterozygous SURF1(-/+) and wild type littermates. Surprisingly, no obvious COX deficiency was detected in SURF1(-/-) tissues, although histochemical analysis revealed the presence of COX deficiency in jejunum villi and total mRNA sequencing (RNAseq) showed that several COX subunit-encoding genes were significantly down regulated in SURF1(-/-) skeletal muscles. In addition, neuropathological findings, indicated a delay in central nervous system development of newborn SURF1(-/-) piglets. Our results suggest a broader role of sSURF1 in mitochondrial bioenergetics.
Genome editing
Leigh syndrome
Mitochondrial disease
Pig
SURF1 KO
Animals
Animals, Genetically Modified
Animals, Newborn
Behavior, Animal
CRISPR-Cas Systems
Cells, Cultured
Central Nervous System
Down-Regulation
Electron Transport Complex IV
Female
Fibroblasts
Gene Editing
Gene Knockout Techniques
Humans
Jejunum
Leigh Disease
Male
Membrane Proteins
Mitochondria
Mitochondrial Proteins
Muscle, Skeletal
Nuclear Transfer Techniques
Primary Cell Culture
Sus scrofa
Disease Models, Animal
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1030672
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