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The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease.

Spermatogonial stem cell transplantation (SSCT) is an experimental technique for transfer of germline between donor and recipient males that could be used as a tool for biomedical research, preservation of endangered species, and dissemination of desirable genetics in food animal populations. To fully realize these potentials, recipient males must be devoid of endogenous germline but possess normal testicular architecture and somatic cell function capable of supporting allogeneic donor stem cell engraftment and regeneration of spermatogenesis. Here we show that male mice, pigs, goats, and cattle harboring knockout alleles of the NANOS2 gene generated by CRISPR-Cas9 editing have testes that are germline ablated but otherwise structurally normal. In adult pigs and goats, SSCT with allogeneic donor stem cells led to sustained donor-derived spermatogenesis. With prepubertal mice, allogeneic SSCT resulted in attainment of natural fertility. Collectively, these advancements represent a major step toward realizing the enormous potential of surrogate sires as a tool for dissemination and regeneration of germplasm in all mammalian species.

Knowledge of the molecular mechanisms by which skeletal muscle hypertrophies in response to increased mechanical loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. To gain insight into potential early signaling mechanisms associated with skeletal muscle hypertrophy, the temporal pattern of mitogen-activated protein kinase (MAPK) phosphorylation and phosphatidylinositol 3-kinase (PI3-kinase) activity during the first 24 h of muscle overload was determined in the rat slow-twitch soleus and fast-twitch plantaris muscles after ablation of the gastrocnemius muscle. p38alpha MAPK phosphorylation was elevated for the entire 24-h overload period in both muscles. In contrast, Erk 2 and p54 JNK phosphorylation were transiently increased by overload, returning to the levels of sham-operated controls by 24 h. PI3-kinase activity was increased by muscle overload only at 12 h of overload and only in the plantaris muscle. In summary, sustained elevation of p38alpha MAPK phosphorylation occurred early in response to muscle overload, identifying this pathway as a potential candidate for mediating early hypertrophic signals in response to skeletal muscle overload.

Pharmacological inhibition of reactive oxygen species (ROS) is a potential strategy to prevent diabetes-induced cardiac dysfunction. This study was designed to investigate precise effects of antioxidant N‑acetylcysteine (NAC) in alleviating diabetic cardiomyopathy (DCM). Echocardiography and histologic studies were performed 12 weeks after streptozocin injection. Protein levels involved in endoplasmic reticulum stress (ERS) and apoptosis were analyzed by western blotting in diabetic hearts or high-glucose (HG, 30 mM)- and palmitic acid (PA, 300 μM)-cultured neonatal rat cardiomyocytes (NRCMs). ROS generation and structural alterations of mitochondria were also assessed. We report that NAC alleviated diabetes-induced cardiac abnormality, including restored ejection fraction (EF %), fraction shortening (FS %), peak E to peak A ratio (E/A) and reduced cardiac hypertrophy and fibrosis. These effects were concomitant with blocked ERS and apoptosis, as evidenced by inactivation of phosphorylated inositol-requiring enzyme-1α (IRE1α)/spliced X-box binding protein 1 (XBP1), phosphorylated protein kinase-like kinase (PERK)/phosphorylated eukaryotic initiation factor 2α (eIF2α) and glucose-regulated protein 78 (GRP78)/activating transcription factor 6 (ATF6α)/C/EBP homologous protein (CHOP) pathways, as well as suppressed Bcl-2-associated X protein (BAX)/B-cell lymphoma-2 (Bcl-2) and cleaved caspase 3 expressions. Mechanistically, PA mediated excessive mitochondrial ROS generation and oxidative stress, which were antagonized by NAC and Mito-TEMPO, a mitochondrial ROS inhibitor. No effects were noted by addition of apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, and NADPH oxidase 4 (NOX 4) and NOX 2 expressions were not altered, indicating that PA-induced ROS generation is independent of NADPH oxidases. Most intriguingly, HG failed to promote ROS production despite its ability to promote ERS and apoptosis in NRCMs. Collectively, these findings indicate that NAC primarily abrogates PA-mediated mitochondrial ROS through ERS and therefore alleviates myocardial apoptosis but has little effect on HG-induced cardiac injury. This uncovers a potential role for NAC in formulating novel cardioprotective strategies in DCM patients.

Statins are the most effective drugs used in the reduction of intracellular synthesis of cholesterol. Numerous studies have confirmed that statins reduce the risk of multiple types of cancers. Statin use in cancer patients is associated with reduced cancer-related mortality. Epithelial-to-mesenchymal transition (EMT), a complicated process programmed by multiple genes, is an important mechanism of cancer metastasis. We explored the effect and mechanism of atorvastatin on the EMT process in A549 cells by establishing an EMT model in vitro induced by TGF-β1, and evaluated the effects of atorvastatin on the lower signaling pathway of TGF-β1 stimulation. Our results showed that atorvastatin partially inhibited the EMT process, and inhibited cell migration and actin filament remodeling. Transcriptional upregulation of ZEB1 and protein sphingosine kinase 1 (SphK1) induced by TGF-β1 was also suppressed. SphK1 plasmid transient transfection strengthened the EMT process induced by TGF-β1 in the presence of atorvastatin. Our experiments confirmed that atorvastatin can partially inhibit the EMT process of non-small cell lung cancer cells induced by TGF-β1 by attenuating the upregulation of SphK1.

Background Circular RNAs (circRNAs) are important regulators in the pathogenesis of diseases and affects the occurrence and development of diseases. However, the role of circRNAs in osteosarcoma (OS) has not been fully elucidated. Methods The expression of circ_0000285, miR-409-3p and insulin-like growth factor binding protein 3 (IGFBP3) was detected using quantitative real-time PCR (qRT-PCR). The protein level of IGFBP3 was measured using western blot. CCK-8 and colony formation assays were used to determine cell proliferation. Flow cytometry was applied to measure cell cycle and cell apoptosis. Transwell assay was used to assess cell invasion and migration. Dual-luciferase reporter assay and RNA Binding Protein Immunoprecipitation (RIP) assay were performed to determine the relationship among circ_0000285, miR-409-3p and IGFBP3. The animal experiments were performed to determine the function of circ_0000285 in vivo. Results In this study, we found that the expression of circ_0000285 was significantly increased in OS tissues and cells and was enriched in the cytoplasm. Knockdown of circ_0000285 inhibited OS growth in vitro and in vivo. Moreover, miR-409-3p was a target miRNA of circ_0000285 and miR-409-3p targets to IGFBP3 in OS. Besides, circ_0000285 could promote proliferation, migration, invasion and inhibit apoptosis of osteosarcoma by miR-409-3p/IGFBP3 axis. Conclusion In this study, circ_0000285 regulated proliferation, migration, invasion and apoptosis of OS cells by miR-409-3p/IGFBP3 axis, implying that circ_0000285 was a potential target for OS therapy.

Accelerated development of novel CRISPR/Cas9-based genome editing techniques provides a feasible approach to introduce a variety of precise modifications in the mammalian genome, including introduction of multiple edits simultaneously, efficient insertion of long DNA sequences into specific targeted loci as well as performing nucleotide transitions and transversions. Thus, the CRISPR/Cas9 tool has become the method of choice for introducing genome alterations in livestock species. The list of new CRISPR/Cas9-based genome editing tools is constantly expanding. Here, we discuss the methods developed to improve efficiency and specificity of gene editing tools as well as approaches that can be employed for gene regulation, base editing, and epigenetic modifications. Additionally, advantages and disadvantages of two primary methods used for the production of gene-edited farm animals: somatic cell nuclear transfer (SCNT or cloning) and zygote manipulations will be discussed. Furthermore, we will review agricultural and biomedical applications of gene editing technology.