Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets.

Myostatin (MSTN) is a negative regulator of myogenesis, and disruption of its function causes increased muscle mass in various species. Here, we report the generation of MSTN-knockout (KO) pigs using genome editing technology combined with somatic-cell nuclear transfer (SCNT). Transcription activator-like effector nuclease (TALEN) with non-repeat-variable di-residue variations, called Platinum TALEN, was highly efficient in modifying genes in porcine somatic cells, which were then used for SCNT to create MSTN KO piglets. These piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibers. These results demonstrate that loss of MSTN increases muscle mass in pigs, which may help increase pork production for consumption in the future.

AMPK activation by AICAR inhibits myogenic differentiation and myostatin expression in cattle.

AMP-activated protein kinase (AMPK) regulates metabolism in skeletal muscle, and myostatin (MSTN) negatively regulates skeletal muscle development and growth. In the present study, AMPK activation and the relationship between AMPK and MSTN during myogenic differentiation were investigated in cultures derived from bovine skeletal muscle. Myoblasts capable of forming myotubes were obtained from bovine skeletal muscle and treated with AICAR to activate AMPK, resulting in suppressed myotube formation. AICAR treatment significantly reduced the expression of MSTN mRNA during myogenic differentiation. Combined treatment with AICAR and MSTN suppressed myotube formation to a greater extent than AICAR alone. SB431542, an inhibitor of MSTN signaling, promoted myotube formation during myogenic differentiation. However, simultaneous treatment with AICAR blocked this effect of SB431542. Therefore, AMPK activation inhibits myogenic differentiation but may suppress MSTN expression to balance muscle development.

Somatic cell reprogramming-free generation of genetically modified pigs.

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

Antigen exposure in the late light period induces severe symptoms of food allergy in an OVA-allergic mouse model.

The mammalian circadian clock controls many physiological processes that include immune responses and allergic reactions. Several studies have investigated the circadian regulation of intestinal permeability and tight junctions known to be affected by cytokines. However, the contribution of circadian clock to food allergy symptoms remains unclear. Therefore, we investigated the role of the circadian clock in determining the severity of food allergies. We prepared an ovalbumin food allergy mouse model, and orally administered ovalbumin either late in the light or late in the dark period under light-dark cycle. The light period group showed higher allergic diarrhea and weight loss than the dark period group. The production of type 2 cytokines, IL-13 and IL-5, from the mesenteric lymph nodes and ovalbumin absorption was higher in the light period group than in the dark period group. Compared to the dark period group, the mRNA expression levels of the tight junction proteins were lower in the light period group. We have demonstrated that increased production of type 2 cytokines and intestinal permeability in the light period induced severe food allergy symptoms. Our results suggest that the time of food antigen intake might affect the determination of the severity of food allergy symptoms.

Expression of myostatin in neural cells of the olfactory system.

Recent studies show that myostatin mRNA expression is found in some regions of the brain. However, the functional significance of this is currently unknown. We therefore investigated myostatin expression and function in the brain. In this study, we used immunohistochemistry, in situ hybridization, and RT-PCR analysis to reveal that myostatin is expressed in the mitral cells in the olfactory bulb (OB) and in neurons in the olfactory cortex (OC). Using 3D reconstruction, mitral cells positive for myostatin were positioned in the lateral and ventral regions of the OB. In contrast, myostatin-positive mitral cells were detected in mice at 2 weeks of age, but not on days 0 and 7 after birth. Activin receptor IIB, a myostatin receptor, was expressed in the OB, OC, hippocampus, and paraventricular thalamic nucleus. Moreover, c-Fos immunostaining in granule cells in the OB was augmented after intracerebroventricular injection of myostatin. These findings suggest that myostatin is localized in specific cells associated with the olfactory system of the brain and may act as a key inhibitor in cell and/or signal development of the olfactory system.