Many studies have been conducted to improve economically important livestock traits such as feed efficiency and muscle growth. Genome editing technologies represent a major advancement for both basic research and agronomic biotechnology development. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technical platform is a powerful tool used to engineer specific targeted loci. However, the potential occurrence of off‐target effects, including the cleavage of unintended targets, limits the practical applications of Cas9‐mediated genome editing. In this study, to minimize the off‐target effects of this technology, we utilized D10A‐Cas9 nickase to generate myostatin‐knockout (MSTN KO) chickens via primordial germ cells. D10A‐Cas9 nickase (Cas9n)‐mediated MSTN KO chickens exhibited significantly larger skeletal muscles in the breast and leg. Degrees of skeletal muscle hypertrophy and hyperplasia induced by myostatin deletion differed by sex and muscle type. The abdominal fat deposition was dramatically lower in MSTN KO chickens than in wild‐type chickens. Our results demonstrate that the D10A‐Cas9 technical platform can facilitate precise and efficient targeted genome engineering and may broaden the range of applications for genome‐edited chickens in practical industrialization and as animal models of human diseases.

ObjectiveIn the poultry industry, the most important economic traits are meat quality and carcass yield. Thus, many studies were conducted to investigate the regulatory pathways during muscle differentiation. To gain insight of muscle differentiation mechanism during growth period, we identified and validated calcium-related genes which were highly expressed during muscle differentiation through mRNA sequencing analysis.MethodsWe conducted next-generation-sequencing (NGS) analysis of mRNA from undifferentiated QM7 cells and differentiated QM7 cells (day 1 to day 3 of differentiation periods). Subsequently, we obtained calcium related genes related to muscle differentiation process and examined the expression patterns by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).ResultsThrough RNA sequencing analysis, we found that the transcription levels of six genes (troponin C1, slow skeletal and cardiac type [TNNC1], myosin light chain 1 [MYL1], MYL3, phospholamban [PLN], caveolin 3 [CAV3], and calsequestrin 2 [CASQ2]) particularly related to calcium regulation were gradually increased according to days of myotube differentiation. Subsequently, we validated the expression patterns of calcium-related genes in quail myoblasts. These results indicated that TNNC1, MYL1, MYL3, PLN, CAV3, CASQ2 responded to differentiation and growth performance in quail muscle.ConclusionThese results indicated that calcium regulation might play a critical role in muscle differentiation. Thus, these findings suggest that further studies would be warranted to investigate the role of calcium ion in muscle differentiation and could provide a useful biomarker for muscle differentiation and growth.

Background: In the poultry and livestock industries, precise genetic information is crucial for improving economic traits. Thus, functional genomic studies help to generate faster, healthier, and more efficient animal production. Chicken myoblast cells, which are required for muscle development and regeneration, are particularly important because chicken growth is closely related to muscle mass. Results: In this study, we induced expression of microRNA-146b-5p mediated by the piggyBac transposon system in primary chicken myoblast (pCM) cells. Subsequently, we analyzed and compared the proliferation and differentiation capacity and also examined the expression of related genes in regular pCM (rpCM) cells and pCM cells overexpressing miRNA-146b-5p (pCM-146b OE cells). pCM-146b OE cells showed increased proliferation and upregulated gene expression related to cell proliferation. In addition, next-generation sequencing analyses were performed to compare global gene expression patterns between rpCM cells and pCM-146b OE cells. We found that the higher proliferation in pCM-146b OE cells was the result of upregulation of gene sets related to the cell cycle. Moreover, miRNA-146b-5p overexpression had inhibitory effects on myotube differentiation in pCM cells. Conclusions: Collectively these results demonstrate that miR-146b-5p is closely related to the proliferation and differentiation of chicken myogenic cells as a modulator of post-transcription. Background Since the genome sequences of avian species have become available, research has aimed to increase muscle mass, enhance muscle regeneration, and reduce fatty acid accumulation to improve growth. Understanding the genes or genetic markers involved in biological functions and regulatory pathways can help to improve economically important traits in the poultry industry [1-3]. Thus, functional genomic studies are powerful and effective for investigating the modulatory mechanisms between cell proliferation and differentiation, in particular in skeletal muscles [4-7]. Our study was conducted in chicken myoblasts derived from embryonic tissue. Myoblasts are derived from satellite cells, which are a precursor to myogenesis [8]. In the quiescent satellite cell stage, PAX7, a critical marker of undifferentiated myoblasts, is highly expressed. After activation, myoblasts start to proliferate, decrease expression of PAX7, and increase expression of MYOD, a myogenic regulatory factor (MRF). Then they enter the stage of terminal differentiation. In this stage, expression of MYOD decreases, and expression of markers of terminal differentiation such as Myogenin and Desmin increases. Eventually myoblast cells form new myotubes, which in turn form new myofibers [9]. Therefore, myoblasts are closely related to muscle growth, which is an economically important trait of domestic animals.

Objective Although previous studies have reported impaired performance in the reading the mind in the eyes test (RMET), which measures complex emotion recognition abilities, in patients with schizophrenia, reports regarding individuals at clinical high risk (CHR) for psychosis have been inconsistent, mainly due to the interacting confounding effects of general cognitive abilities and age. We compared RMET performances across first-episode psychosis (FEP) patients, CHR individuals, and healthy controls (HCs) while controlling for the effects of both general cognitive abilities and age.Methods A total of 25 FEP, 41 CHR, and 44 HC subjects matched for age participated in this study. RMET performance scores were compared across the groups using analysis of variance with sex and intelligence quotient as covariates. Exploratory Pearson’s correlation analyses were performed to reveal the potential relationships of RMET scores with clinical symptom severity in the FEP and CHR groups.Results RMET performance scores were significantly lower among FEP and CHR participants than among HCs. FEP patients and CHR subjects showed comparable RMET performance scores. RMET scores were negatively correlated with Positive and Negative Syndrome Scale (PANSS) positive symptom subscale scores in the FEP patients. No significant correlation was identified between RMET scores and other clinical scale scores.Conclusion Impaired RMET performance is present from the risk stage of psychosis, which might be related to positive symptom severity in early psychosis. Longitudinal studies are necessary to confirm the stability of complex emotion recognition impairments and their relationship with social functioning in early psychosis patients.

A bioreactor can be used for mass production of therapeutic proteins and other bioactive substances. Although various methods have been developed using microorganisms and animal cells, advanced strategies are needed for the efficient production of biofunctional proteins. In microorganisms, post-translational glycosylation and modification are not performed properly, while animal cell systems require more time and expense. To overcome these problems, new methods using products from transgenic animals have been considered, such as genetically modified cow’s milk and hen’s eggs. In this study, based on a non-viral piggyBac transposition system, we generated transgenic bioreactor chickens that produced human cystatin C (hCST3). There were no differences in the phenotype or histochemical structure of the wild-type and hCST3-expressing transgenic chickens. Subsequently, we analyzed the hCST3 expression in transgenic chickens, mainly in muscle and egg white, which could be major deposition warehouses for hCST3 protein. In both muscle and egg white, we detected high hCST3 expression by ELISA and Western blotting. hCST3 proteins were efficiently purified from muscle and egg white of transgenic chickens using a His-tag purification system. These data show that transgenic chickens can be efficiently used as a bioreactor for the mass production of bioactive materials.