Extracellular vesicles from induced pluripotent stem cell-derived mesenchymal stem cells enhance the recovery of acute kidney injury.

BACKGROUND AIMS: To investigate whether the extracellular vesicles (EVs) from mesenchymal stem cell-like cells derived from induced pluripotent stem cells (iMSC-EVs) can inhibit the progression of acute kidney injury (AKI). METHODS: The characteristics of iMSC-EVs were confirmed by immunoblotting, cryo-transmission electron microscopy, nanoparticle tracking analysis, and their localization in kidneys. Using human renal epithelial cells, the potential of iMSC-EVs to stimulate the growth and survival of HK-2 cells undergoing cisplatin-induced cell death was investigated. The anti-inflammatory effects of iMSC-EVs was examined in M1-polarized THP-1 macrophages. Subsequently, the therapeutic potential of iMSC-EVs was assessed in cisplatin-induced acute kidney injury in BALB/c mice. The anti-apoptotic and anti-inflammatory effect of iMSC-EVs was evaluated using serum biochemistry, histology, immunohistochemistry, and gene expression analysis. RESULTS: iMSC-EVs promoted the growth of renal epithelial cell (HK-2) and enhanced the survival of HK-2 undergoing cisplatin-induced cell death. In cisplatin-induced mice with AKI, iMSC-EVs alleviated AKI, as shown by reduced blood nitrogen urea/creatinine and increased body weight. Also, iMSC-EVs enhanced renal tissue integrity and the number of proliferating cell nuclear antigen-positive tubules. iMSC-EVs decreased the infiltration of immune cells, reduced the expression of inflammatory genes in M1-induced THP-1 cells and enhanced capillary density in the kidney of AKI mice. Real-time quantitative polymerase chain reaction analysis showed that the expression of inflammatory genes in the kidney of AKI mice was reduced compared with that received vehicle. Immunoblotting revealed that iMSC-EVs led to a decreased protein expression of key inflammatory genes. Also, iMSC-EVs reversed the activation of ERK1/2 signaling induced by AKI. Finally, iMSC-EVs inhibited the apoptosis of HK-2 cells induced by cisplatin as well as that of renal tissue of AKI mice. CONCLUSIONS: Our data suggest that iMSC-EVs have potential to become a novel, cell-free therapeutic for cisplatin-induced AKI.

Aberrant glucose metabolism underlies impaired macrophage differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that is responsible for transporting G6P into the endoplasmic reticulum. GSD-Ib is characterized by disturbances in glucose homeostasis, neutropenia, and neutrophil dysfunction. Although some studies have explored neutrophils abnormalities in GSD-Ib, investigations regarding monocytes/macrophages remain limited so far. In this study, we examined the impact of G6PT deficiency on monocyte-to-macrophage differentiation using bone marrow-derived monocytes from G6pt-/- mice as well as G6PT-deficient human THP-1 monocytes. Our findings revealed that G6PT-deficient monocytes exhibited immature differentiation into macrophages. Notably, the impaired differentiation observed in G6PT-deficient monocytes seemed to be associated with abnormal glucose metabolism, characterized by enhanced glucose consumption through glycolysis, even under quiescent conditions with oxidative phosphorylation. Furthermore, we observed a reduced secretion of inflammatory cytokines in G6PT-deficient THP-1 monocytes during the inflammatory response, despite their elevated glucose consumption. In conclusion, this study sheds light on the significance of G6PT in monocyte-to-macrophage differentiation and underscores its importance in maintaining glucose homeostasis and supporting immune response in GSD-Ib. These findings may contribute to a better understanding of the pathogenesis of GSD-Ib and potentially pave the way for the development of targeted therapeutic interventions.

Molecular mechanisms of aberrant neutrophil differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is caused by a deficiency in glucose-6-phosphate transporter (G6PT). Neutropenia in GSD-Ib has been known to result from enhanced apoptosis of neutrophils. However, it has also been raised that neutrophil maturation arrest in the bone marrow would contribute to neutropenia. We now show that G6pt-/- mice exhibit severe neutropenia and impaired neutrophil differentiation in the bone marrow. To investigate the role of G6PT in myeloid progenitor cells, the G6PT gene was mutated using CRISPR/Cas9 system, and single cell-derived G6PT-/- human promyelocyte HL-60 cell lines were established. The G6PT-/- HL-60s exhibited impaired neutrophil differentiation, which is associated with two mechanisms: (i) abnormal lipid metabolism causing a delayed metabolic reprogramming and (ii) reduced nuclear transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ) in G6PT-/- HL-60s. In this study, we demonstrated that G6PT is essential for neutrophil differentiation of myeloid progenitor cells and regulates PPARγ activity.

Prokineticin receptor 1 ameliorates insulin resistance in skeletal muscle.

Type 2 diabetes mellitus may result from insulin resistance in skeletal muscle. Prokineticin receptor 1 (Prokr1) improves metabolic phenotype in adipose tissue and the cardiovascular system; however, its effects on skeletal muscle have not been investigated. We investigated the Prokr1 signaling pathways and its metabolic function in murine myoblast, satellite cells, and their differentiated myotubes. We measured the expression levels of Prokr1 in the skeletal muscle of mice as well as human skeletal muscle cell-derived myotubes. Prokineticin 2 (PROK2), a ligand of PROKR1, induced calcium mobilization in a dose-dependent manner and altered the mRNA levels of 578 genes in PROKR1-overexpressed HEK293T cells. Functional enrichment of differentially expressed genes revealed that PROKR1 activated Gq-mediated PI3K/AKT and MAPK/ERK signaling pathways in skeletal muscle cells. Prokr1 significantly activated the PI3K/AKT signaling pathway in myotubes derived from C2C12 and satellite cells, regardless of the presence or absence of insulin. Prokr1 also promoted the translocation of glucose transporter 4 (GLUT4) into the plasma membrane. In palmitate-induced insulin-resistant myotubes, Prokr1 enhanced insulin-stimulated AKT phosphorylation, GLUT4 translocation, and glucose uptake. mRNA and protein levels of Prokr1 were significantly decreased in skeletal muscle and white adipose tissue of diet-induced obese mice, and the amount of PROKR1 protein was significantly decreased in human skeletal muscle cell-derived myotubes under insulin resistance conditions. Taken together, these results demonstrate that Prokr1 plays an important role in insulin sensitivity and is a potential therapeutic target to ameliorate insulin resistance in skeletal muscle.

Generation of myostatin-knockout chickens mediated by D10A-Cas9 nickase.

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.

PROKR1 delivery by cell-derived vesicles restores the myogenic potential of Prokr1-deficient C2C12 myoblasts.

Cell-derived vesicles (CDVs) have been investigated as an alternative to exosomes. Here, we generated CDVs from Prokineticin receptor 1 (PROKR1) overexpressing HEK293T cells using micro-extrusion. More than 60 billion PROKR1-enriched CDV (PROKR1Tg CDVs) particles with canonical exosome properties were recovered from 107 cells. With 25 µg/mL of PROKR1Tg CDVs, we observed delivery of PROKR1, significant reduction of apoptosis, and myotube formation in C2C12Prokr1-/- myoblasts that have lost their myogenic potential but underwent apoptosis following myogenic commitment. Expression levels of early and late myogenic marker genes and glucose uptake capacity were restored to equivalent levels with wild-type control. Furthermore, PROKR1Tg CDVs were accumulated in soleus muscle comparable to the liver without significant differences. Therefore, CDVs obtained from genetically engineered cells appear to be an effective method of PROKR1 protein delivery and offer promise as an alternative therapy for muscular dystrophy.

Glucose-6-phosphate transporter mediates macrophage proliferation and functions by regulating glycolysis and mitochondrial respiration.

Glycogen storage disease type Ib (GSD-Ib), caused by a deficiency in glucose-6-phosphate transporter (G6PT), is characterized by disrupted glucose homeostasis, inflammatory bowel disease, neutropenia, and neutrophil dysfunction. The purpose of this study was to investigate the role of G6PT on macrophage functions and metabolism. Peritoneal macrophages of G6pt-/- mice were lower in number and their effector functions including migration, superoxide production, and phagocytosis were impaired. To investigate the underlying mechanisms of macrophage dysfunction, the G6PT gene was mutated in porcine alveolar macrophage 3D4/31 cells using the CRISPR/Cas9 technology. The G6PT-deficient macrophages exhibited significant decline in cell growth, bactericidal activity, and antiviral response. These phenotypes are associated with the impaired glycolysis and mitochondrial oxidative phosphorylation. We therefore propose that the G6PT-mediated metabolism is essential for effector functions of macrophage, the immune deficiencies observed in GSD-Ib extend beyond neutropenia and neutrophil dysfunction, and future therapeutic targets aimed both the neutrophils and macrophages may be necessary.

Uterine epithelial expression of the tumor necrosis factor superfamily: a strategy for immune privilege during pregnancy in a true epitheliochorial placentation species.

The maternal immune system tolerates semi-allogeneic placental tissues during pregnancy. Fas ligand (FASLG) and tumor necrosis factor superfamily 10 (TNFSF10) are known to be components of maternal immune tolerance in humans and mice. However, the role of FASLG and TNFSF10 in the tolerance process has not been studied in pigs, which form a true epitheliochorial type placenta. Thus, the present study examined the expression and function of FASLG and TNFSF10 and their receptors at the maternal-conceptus interface in pigs. The endometrium and conceptus tissues expressed FASLG and TNFSF10 and their receptor mRNAs during pregnancy in a stage-specific manner. During pregnancy, FASLG and TNFSF10 proteins were localized predominantly to endometrial luminal epithelial cells with strong signals on Day 30 to term and on Day 15, respectively, and receptors for TNFSF10 were localized to some stromal cells. Interferon-γ (IFNG) increased the expression of TNFSF10 and FAS in endometrial tissues. Co-culture of porcine endometrial epithelial cells over-expressing TNFSF10 with peripheral blood mononuclear cells yielded increased apoptotic cell death of lymphocytes and myeloid cells. In addition, many apoptotic T cells were found in the endometrium on Day 15 of pregnancy. The present study demonstrated that FASLG and TNFSF10 were expressed at the maternal-conceptus interface and conceptus-derived IFNG increased endometrial epithelial TNFSF10, which, in turn, induced apoptotic cell death of immune cells. These results suggest that endometrial epithelial FASLG and TNFSF10 may be critical for the formation of micro-environmental immune privilege at the maternal-conceptus interface for the establishment and maintenance of pregnancy in pigs.

Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken.

Chicken as a food source is one of the most widespread domestic animals, and it has been used extensively as a research model. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system is the most efficient and reliable tool for precise genome-targeted modification and has generated considerable excitement for industrial applications, as well as biologic science. Unlike in mammals, germline-transmittable primordial germ cells (PGCs) in chicken were used as an alternative strategy for the production of genetically altered chickens. Here, by combining the CRISPR-Cas9 platform and germ cell-mediated germline transmission, we generated G0/G1 switch gene 2 ( G0S2) knockout (KO) chickens, and G0S2 null KO chickens showed a dramatic reduction of abdominal fat deposition without affecting other economic traits. Additionally, G0S2 null KO chickens had altered fatty acid compositions in their blood and abdominal fat compared with wild-type chickens under normal dietary conditions. The global mRNA sequencing data showed that G0S2 disruption in chickens would activate the adipose tissue-specific peroxisomal oxidation pathway, and enoyl-coenzyme A (CoA), hydratase/3-hydroxyacyl CoA dehydrogenase might be a target molecule in metabolic homeostasis in the chicken adipose tissue. Our results demonstrate that the CRISPR-Cas9 system with chicken PGCs can facilitate the production of specific genome-edited chickens for practical applications, as well as basic research.-Park, T. S., Park, J., Lee, J. H., Park, J.-W., Park, B.-C. Disruption of G0/G1 switch gene 2 ( G0S2) reduced abdominal fat deposition and altered fatty acid composition in chicken.

Characterization of splenic MRC1hiMHCIIlo and MRC1loMHCIIhi cells from the monocyte/macrophage lineage of White Leghorn chickens.

Monocytes/macrophages, which are found in a variety of organs, maintain tissue homeostasis at a steady state and act as the first line of defence during pathogen-induced inflammation in the host. Most monocyte/macrophage lineage studies in chickens have been largely performed using cell lines, while few studies using primary cells have been conducted. In the present study, the phenotypic and functional characteristics of splenic monocyte/macrophage lineage cells during steady state and inflammatory conditions were examined. Splenic monocyte/macrophage lineage cells could be identified as MRC1loMHCIIhi and MRC1hiMHCIIlo cells based on their surface expression of MRC1 and MHCII. In the steady state, MRC1loMHCIIhi cells were more frequently found among MRC1+ cells. MRC1loMHCIIhi cells expressed a higher number of antigen-presenting molecules (MHCII, MHCI, and CD80) than MRC1hiMHCIIlo cells. In contrast, MRC1hiMHCIIlo cells showed better phagocytic and CCR5-dependent migratory properties than MRC1loMHCIIhi cells. Furthermore, MRC1hiMHCIIlo cells infiltrated the spleen in vivo and then became MRC1loMHCIIhi cells. During lipopolysaccharide (LPS)-induced inflammatory conditions that were produced via intraperitoneal (i.p.) injection, the proportion and absolute number of MRC1hiMHCIIlo cells were increased in the spleen. Uniquely, inflammation induced the downregulation of MHCII expression in MRC1hiMHCIIlo cells. The major source of inflammatory cytokines (IL-1ß, IL-6, and IL-12) was MRC1loMHCIIhi cells. Furthermore, MRC1hiMHCIIlo cells showed greater bactericidal activity than MRC1loMHCIIhi cells during LPS-induced inflammation. Collectively, these results suggest that two subsets of monocyte/macrophage lineage cells exist in the chicken spleen that have functional differences.

Bacillus subtilis spores as adjuvants against avian influenza H9N2 induce antigen-specific antibody and T cell responses in White Leghorn chickens.

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1ß and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4+ and CD8+ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4+ and CD8+ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Muscle differentiation induced by p53 signaling pathway-related genes in myostatin-knockout quail myoblasts.

The myostatin (MSTN) gene is of interest in the livestock industry because mutations in this gene are closely related to growth performance and muscle differentiation. Thus, in this study, we established MSTN knockout (KO) quail myoblasts (QM7) and investigated the regulatory pathway of the myogenic differentiation process. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate MSTN KO QM7 cells and subsequently isolated a single cell-derived MSTN KO QM7 subline with 10- and 16-nucleotide deletions that induced translational frameshift mutations. The differentiation capacity and proliferation rate of MSTN KO QM7 cells were enhanced. We conducted next-generation-sequencing (NGS) analysis to compare the global gene expression profiles of wild-type (WT) QM7 and MSTN KO QM7 cells. Intriguingly, NGS expression profiles showed different expression patterns of p21 and p53 in MSTN KO QM7 cells. Moreover, we identified downregulated expression patterns of leukemia inhibitory factor and DNA Damage Inducible Transcript 4, which are genes in the p53 signaling pathway. Using quantitative RT-PCR (qRT-PCR) analysis and western blotting, we concluded that p53-related genes promote the cell cycle by upregulating p21 and enhancing muscle differentiation in MSTN KO QM7 cells. These results could be applied to improve economic traits in commercial poultry by regulating MSTN-related networks.

High Homology-Directed Repair Using Mitosis Phase and Nucleus Localizing Signal.

In homology-directed repair, mediated knock-in single-stranded oligodeoxynucleotides (ssODNs) can be used as a homologous template and present high efficiency, but there is still a need to improve efficiency. Previous studies have mainly focused on controlling double-stranded break size, ssODN stability, and the DNA repair cycle. Nevertheless, there is a lack of research on the correlation between the cell cycle and single-strand template repair (SSTR) efficiency. Here, we investigated the relationship between cell cycle and SSTR efficiency. We found higher SSTR efficiency during mitosis, especially in the metaphase and anaphase. A Cas9 protein with a nuclear localization signal (NLS) readily migrated to the nucleus; however, the nuclear envelope inhibited the nuclear import of many nucleotide templates. This seemed to result in non-homologous end joining (NHEJ) before the arrival of the homologous template. Thus, we assessed whether NLS-tagged ssODNs and free NLS peptides could circumvent problems posed by the nuclear envelope. NLS-tagging ssODNs enhanced SSTR and indel efficiency by 4-fold compared to the control. Our results suggest the following: (1) mitosis is the optimal phase for SSTR, (2) the donor template needs to be delivered to the nucleus before nuclease delivery, and (3) NLS-tagging ssODNs improve SSTR efficiency, especially high in mitosis.

Effects of Angelica gigas Nakai on the production of decursin- and decursinol angelate-enriched eggs.

BACKGROUND: The livestock industry requires high-quality products, as well as improved productivity. There have been many studies regarding the utilization of feed additives aiming to increase productivity, enhance immune functions and prevent infectious diseases in livestock. Biofunctional feed additives would be beneficial not only for animal health, but also for consumers. In the present study, we utilized root and byproduct (stem and leaf) powders of Angelica gigas Nakai (AGN, Korean Danggui) as feed additives and examined the deposition of biofunctional compounds, such as decursin and decursinol angelate, into egg white and yolk. RESULTS: We optimized the detection system for decursin and decursinol angelate, and determined the amounts of decursin and decursinol angelate derived from AGN byproducts (stem and leaf) as well as root. In Experiment 1, laying hens were fed with the dried AGN root powder and the effective compounds were detected in egg white and yolk. Subsequently, in Experiment 2, we examined AGN byproducts as an alternative feeding supplement. Additionally, biochemical parameters were analyzed to evaluate changes in the health of the hens by feeding AGN root powder. The results obtained indicated that decursin and decursinol angelate were stably transferred into egg white and yolk by feeding AGN byproducts as well as root. Intriguingly, plasma cholesterol levels were significantly decreased in a dose-dependent manner, and those of interleukin-1ß, as an immune-related biomarker, were considerably increased in the treated hens. CONCLUSION: These results indicated that AGN root and byproducts (stem and leaf) could be utilized for the production of value-added eggs and improving the health of hens in the poultry industry. © 2018 Society of Chemical Industry.

Effects of exercise on myokine gene expression in horse skeletal muscles.

OBJECTIVE: To examine the regulatory effects of exercise on myokine expression in horse skeletal muscle cells, we compared the expression of several myokine genes (interleukin 6 [IL-6], IL-8, chemokine [C-X-C motif] ligand 2 [CXCL2], and chemokine [C-C motif] ligand 4 [CCL4]) after a single bout of exercise in horses. Furthermore, to establish in vitro systems for the validation of exercise effects, we cultured horse skeletal muscle cells and confirmed the expression of these genes after treatment with hydrogen peroxide. METHODS: The mRNA expression of IL-6, IL-8, CXCL2, and CCL4 after exercise in skeletal muscle tissue was confirmed using quantitative-reverse transcriptase polymerase chain reactions (qRT-PCR). We then extracted horse muscle cells from the skeletal muscle tissue of a neonatal Thoroughbred. Myokine expression after hydrogen peroxide treatments was confirmed using qRT-PCR in horse skeletal muscle cells. RESULTS: IL-6, IL-8, CXCL2, and CCL4 expression in Thoroughbred and Jeju horse skeletal muscles significantly increased after exercise. We stably maintained horse skeletal muscle cells in culture and confirmed the expression of the myogenic marker, myoblast determination protein (MyoD). Moreover, myokine expression was validated using hydrogen peroxide (H2O2)-treated horse skeletal muscle cells. The patterns of myokine expression in muscle cells were found to be similar to those observed in skeletal muscle tissue. CONCLUSION: We confirmed that several myokines involved in inflammation were induced by exercise in horse skeletal muscle tissue. In addition, we successfully cultured horse skeletal muscle cells and established an in vitro system to validate associated gene expression and function. This study will provide a valuable system for studying the function of exercise-related genes in the future.

The effect of heat stress on frame switch splicing of X-box binding protein 1 gene in horse.

OBJECTIVE: Among stress responses, the unfolded protein response (UPR) is a well-known mechanism related to endoplasmic reticulum (ER) stress. ER stress is induced by a variety of external and environmental factors such as starvation, ischemia, hypoxia, oxidative stress, and heat stress. Inositol requiring enzyme 1α (IRE1α)-X-box protein 1 (XBP1) is the most conserved pathway involved in the UPR and is the main component that mediates IRE1α signalling to downstream ER-associated degradation (ERAD)- or UPR-related genes. XBP1 is a transcription factor synthesised via a novel mechanism called 'frame switch splicing', and this process has not yet been studied in the horse XBP1 gene. Therefore, the aim of this study was to confirm the frame switch splicing of horse XBP1 and characterise its dynamics using Thoroughbred muscle cells exposed to heat stress. METHODS: Primary horse muscle cells were used to investigate heat stress-induced frame switch splicing of horse XBP1. Frame switch splicing was confirmed by sequencing analysis. XBP1 amino acid sequences and promoter sequences of various species were aligned to confirm the sequence homology and to find conserved cis-acting elements, respectively. The expression of the potential XBP1 downstream genes were analysed by quantitative real-time polymerase chain reaction. RESULTS: We confirmed that splicing of horse XBP1 mRNA was affected by the duration of thermal stress. Twenty-six nucleotides in the mRNA of XBP1 were deleted after heat stress. The protein sequence and the cis-regulatory elements on the promoter of horse XBP1 are highly conserved among the mammals. Induction of putative downstream genes of horse XBP1 was dependent on the duration of heat stress. We confirmed that both the mechanisms of XBP1 frame switch splicing and various binding elements found in downstream gene promoters are highly evolutionarily conserved. CONCLUSION: The frame switch splicing of horse XBP1 and its dynamics were highly conserved among species. These results facilitate studies of ER-stress in horse.

Muscle differentiation induced up-regulation of calcium-related gene expression in quail myoblasts.

OBJECTIVE: In 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. METHODS: We 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). RESULTS: Through 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. CONCLUSION: These 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.

Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

C-X-C chemokine receptor type 4 (CXCR4) is a key receptor for chicken primordial germ cell migration.

In mammals, germ cells originate outside of the developing gonads and follow a unique migration pattern through the embryonic tissue toward the genital ridges. Many studies have attempted to identify critical receptors and factors involved in germ cell migration. However, relatively few reports exist on germ cell receptors and chemokines that are involved in germ cell migration in avian species. In the present study, we investigated the specific migratory function of C-X-C chemokine receptor type 4 (CXCR4) in chicken primordial germ cells (PGCs). We induced loss-of-function via a frameshift mutation in the CXCR4 gene in chicken PGCs using clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing. The migratory capacity of CXCR4 knockout PGCs was significantly reduced in vivo after transplantation into recipient embryos. However, CXCR4-expressing somatic cell lines, such as chicken DT40 and DF1, failed to migrate into the developing gonads, suggesting that another key factor(s) is necessary for targeting and settlement of PGCs into the genital ridges. In conclusion, we show that CXCR4 plays a critical role in the migration of chicken germ cells.

Targeted gene knockout in chickens mediated by TALENs.

Genetically modified animals are used for industrial applications as well as scientific research, and studies on these animals contribute to a better understanding of biological mechanisms. Gene targeting techniques have been developed to edit specific gene loci in the genome, but the conventional strategy of homologous recombination with a gene-targeted vector has low efficiency and many technical complications. Here, we generated specific gene knockout chickens through the use of transcription activator-like effector nuclease (TALEN)-mediated gene targeting. In this study, we accomplished targeted knockout of the ovalbumin (OV) gene in the chicken primordial germ cells, and OV gene mutant offspring were generated through test-cross analysis. TALENs successfully induced nucleotide deletion mutations of ORF shifts, resulting in loss of chicken OV gene function. Our results demonstrate that the TALEN technique used in the chicken primordial germ cell line is a powerful strategy to create specific genome-edited chickens safely for practical applications.