Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family ) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% ( < 0.01), consequently increasing carcass lean percentage (CLP; < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production.
BackgroundEpigenetic modifications (especially altered DNA methylation) resulting in altered gene expression may be one reason for development failure or abnormalities in cloned animals, but the underlying mechanism of the abnormal phenotype in cloned piglets remains unknown. Some cloned piglets in our study showed abnormal phenotypes such as large tongue (longer and thicker), weak muscles, and exomphalos. Here we conducted DNA methylation (DNAm) immunoprecipitation and high throughput sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) of muscle tissues of cloned piglets to investigate the relationship of abnormal DNAm with gene dysregulation and the unusual phenotypes in cloned piglets.ResultsAnalysis of the methylomes revealed that abnormal cloned piglets suffered more hypomethylation than hypermethylation compared to the normal cloned piglets, although the DNAm level in the CpG Island was higher in the abnormal cloned piglets. Some repetitive elements, such as SINE/tRNA-Glu Satellite/centr also showed differences. We detected 1,711 differentially expressed genes (DEGs) between the two groups, of which 243 genes also changed methylation level in the abnormal cloned piglets. The altered DNA methylation mainly affected the low and silently expressed genes. There were differences in both pathways and genes, such as the MAPK signalling pathway, the hypertrophic cardiomyopathy pathway, and the imprinted gene PLAGL1; all of which may play important roles in development of the abnormal phenotype.ConclusionsThe abnormal cloned piglets showed substantial changes both in the DNAm and the gene expression. Our data may provide new insights into understanding the molecular mechanisms of the reprogramming of genetic information in cloned animals.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-811) contains supplementary material, which is available to authorized users.
Congenital hypothyroidism (CH) is one of the most prevalent endocrine diseases, for which the underlying mechanisms remain unknown; it is often accompanied by anemia and immunodeficiency in patients. Here, we created a severe CH model together with anemia and T lymphopenia to mimic the clinical features of hypothyroid patients by ethylnitrosourea (ENU) mutagenesis in Bama miniature pigs. A novel recessive c.1226A>G transition of the dual oxidase 2 () gene was identified as the causative mutation. This mutation hindered the production of hydrogen peroxide (HO) and thus contributed to thyroid hormone (TH) synthesis failure. Transcriptome sequencing analysis of the thymuses showed that Krüppel-like factor 9 () was predominantly downregulated in hypothyroid mutants. was verified to be directly regulated by TH in a TH receptor (TR)-dependent manner both in vivo and in vitro. Furthermore, knockdown of in zebrafish embryos impaired hematopoietic development including erythroid maturation and T lymphopoiesis. Our findings suggest that the TR-KLF9 axis is responsible for the hematopoietic dysfunction and might be exploited for the development of novel therapeutic interventions for thyroid diseases.
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