Both epidemiologic and experimental animal studies demonstrate that chronic psychological stress exerts adverse effects on the initiation and/or progression of many diseases. However, intergenerational effects of this environmental information remains poorly understood. Here, using a C57BL/6 mouse model of restraint stress, we show that offspring of stressed fathers exhibit hyperglycemia due to enhanced hepatic gluconeogenesis and elevated expression of PEPCK. Mechanistically, we identify an epigenetic alteration at the promoter region of the Sfmbt2 gene, a maternally imprinted polycomb gene, leading to a downregulation of intronic microRNA-466b-3p, which post-transcriptionally inhibits PEPCK expression. Importantly, hyperglycemia in F1 mice is reversed by RU486 treatment in fathers, and dexamethasone administration in F0 mice phenocopies the roles of restraint stress. Thus, we provide evidence showing the effects of paternal psychological stress on the regulation of glucose metabolism in offspring, which may have profound implications for our understanding of health and disease risk inherited from fathers.

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Somatic cell nuclear transfer offers new opportunities for genetic engineering and genome preservation in mammalian animal species. We show that, in addition to cumulus cells, cultured adult rabbit fibroblasts are also capable of supporting full-term development after nuclear transfer. Nuclear transfer embryos constructed using serum-starved fibroblasts showed a significantly higher developmental rate than non-starved fibroblasts through preimplantation stages. A total of 467 nuclear transfer embryos were transferred into the oviducts of pseudo pregnant mothers. Eight of the 20 surrogate rabbits carried the pregnancy to term and five of them gave birth naturally to a total of nine rabbits. However, all of the offspring died before postnatal day 10. A Caesarean section was performed on three surrogates, giving birth to a total of five rabbits, three of them survived and grew into healthy adults. DNA analyses confirmed that these rabbits were genetically identical to the donor male rabbit. The present study demonstrates that rabbits can be cloned from adult fibroblasts after culture.

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Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3'UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA in sheep.

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.

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