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.
Background Many studies have demonstrated that microRNAs, a class of small and non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of miR-592 in the development of obesity-associated metabolic disorders, including hyperglycemia andinsulin resistance. Methods The expression levels of miR-592 were measured in the liver of obese mice and humans by quantitative reverse transcription PCR. Loss- and gain-of function experiments were employed to explore the metabolic function of miR-592 using locked nucleic acids and adenovirus in lean and obese mice, respectively. The molecular target of miR-592 was determined by western blotting and luciferase reporter assays. Findings We found a significant decreased expression of miR-592 in the liver of obese mice and humans. Inhibition of miR-592 led to elevated blood glucose levels, enhanced gluconeogenesis and reduced insulin sensitivity in lean mice. In contrast, adenovirus-mediated overexpression of hepatic miR-592 improved metabolic disorders in obese mice. Mechanistically, we found that the transcription factor forkhead box O1 (FOXO1) is a direct target gene of miR-592 to mediate its metabolic functions. miR-592 was able to inhibit the mRNA and protein expression of FOXO1 by binding to its 3′-untranslated region. Interpretations Our findings demonstrate that obesity-associated down-regulation of miR-592 plays an important role in the progression of metabolic diseases. Restoration of hepatic miR-592 could improve glucose and lipid metabolism in obese mice. Fund This work is supported by the (No. 2016YFC1304805 to Dr. Chen), Natural Science Foundation of China (No. 81771574 to Dr. Wu), Shanghai Science Foundation (No. 18ZR1437800 to Dr. Li), Science and Technology Commission of Shanghai Municipality (Nos.18dz2304400 and 15,411,970,700 to Dr. Yang).
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.
The study was financially supported through grants from the National Key Research Program of China (No. 2016YFC1304800); the National Natural Science Foundation of China (Nos: 81170756, 81571486); the Natural Science Foundation of Shanghai (Nos: 15140901700, 15ZR1424900) and the Programme for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. There are no conflicts of interest to declare.
The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.
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