Myogenic regulatory factors are key players in determining muscle mass and meat quality in Jeju native and Berkshire pigs.

BACKGROUND: Meat from Jeju native pigs (JNPs) is highly popular among Korean consumers; however, the production efficiency is limited due to the low adult body weight. In contrast, the Berkshire breed, which has a genetic background closely related to Asian native pigs, gains weight more efficiently. OBJECTIVES: This study focused on the differential expression of genes related to muscle growth in postnatal myogenesis between Berkshire and JNPs, specifically the myogenic regulatory factor (MRF) genes (MyoD, Pax7, Myf5, Myf6 and MyBPH). The MRF family is primarily involved in the proliferation and development of muscle. METHODS: Qualitative reverse transcription-polymerase chain reaction and western blot analyses revealed that expression of MyoD and Pax7 was significantly higher in Berkshire pigs than in JNPs. In addition, co-expression of MyoD and Pax7 was observed in myotubes formed in cultured C2C12 cells. ToppCluster was used to elucidate the relationship between biological processes of the MRFs and muscle-related signalling pathways. RESULTS: MyoD and Pax7 are factors essential for the activation of satellite cell during myogenesis. However, the mRNA and protein levels of MyBPH (which is responsible for meat quality, e.g. water content, colour and tenderness) are significantly higher in both 1-day-old piglets and adult JNPs than in Berkshire pigs. CONCLUSIONS: This study provides a genetic understanding of myogenesis in the postnatal and adult stages of Berkshire pigs and JNPs. Moreover, these results will help identify marker genes related to muscle mass, growth performance and meat quality in indigenous Korean pig breeds.

The first comprehensive description of the expression profile of genes involved in differential body growth and the immune system of the Jeju Native Pig and miniature pig.

Sus scrofa provides a major source of animal protein for humans as well as being an excellent biomedical model. This study was carried out to understand, in detail, the genetic and functional variants of Jeju Native Pigs and miniature pigs through differential expression profiling of the genes controlling their immune response, growth performance, and meat quality. The Illumina HiSeq 2000 platform was used for generating 1.3 billion 90 bp paired-end reads, which were mapped to the S. scrofa genome using TopHat2. A total of 2481 and 2768 genes were differentially expressed with 8-log changes in muscle and liver samples, respectively. Five hundred forty-eight genes in muscle and 642 genes in liver samples had BLAST matches within the non-redundant database. GO process and pathway analyses showed enhanced biological processes related to the extracellular structural organization and skeletal muscle cell differentiation in muscle tissue, whereas the liver tissue shares functions related to the inflammatory response. Herein, we identify inflammatory regulatory genes in miniature pigs and growth response genes in Jeju Native Pigs, information which can provide a stronger base for the selection of breeding stock and facilitate further in vitro and in vivo studies for therapeutic purposes.

Comparative Transcriptomic Analyses by RNA-seq to Elucidate Differentially Expressed Genes in the Muscle of Korean Thoroughbred Horses.

The athletic abilities of the horse serve as a valuable model to understand the physiology and molecular mechanisms of adaptive responses to exercise. We analyzed differentially expressed genes in triceps brachii muscle tissues collected from Eonjena Taeyang and Jigusang Seryeok Thoroughbred horses and their co-expression networks in a large-scale RNA-sequence dataset comparing expression before and after exercise. High-quality horse transcriptome data were generated, with over 22 million 90-bp pair-end reads. By comparing the annotations, we found that MYH3, MPZ, and PDE8B genes in Eonjena Taeyang and PDE8B and KIF18A genes in Jigusang Seryeok were upregulated before exercise. Notably further, we observed that PPP1R27, NDUFA3, TNC, and ANK1 in Eonjena Taeyang and HIF1A, BDNF, ADRB2, OBSCN, and PER3 in Jigusang Seryeok have shown upregulation at the postexercise period. This investigation suggested that genes responsible for metabolism and oxidative phosphorylations associated with endurance and resistance exercise were highly expressed, whereas genes encoding structural proteins were generally suppressed. The expression profile of racehorses at pre- and postexercise will provide credible reference for further studies on biological effects such as responses to stress and adaption of other Thoroughbred horse, which might be useful for selective breeding for improvement of traits in commercial production.

Characterization and cardiac differentiation of chicken spermatogonial stem cells.

Spermatogonial stem cells (SSCs), a unique population of germline stem cells in adult testis, have the capability to self-renew and produce daughter cells destined to differentiate into spermatozoa throughout the life of the bird. Chicken SSCs were successfully isolated from testicular cells and subsequent analysis was performed to identify pluripotent cells by investigation with cytochemical reagents including Periodic acid-Schiff (PAS), alkaline phosphatase (AP), and antibodies to germline cell specific (DAZL or VASA) and stage-specific embryonic antigens (Oct4, SSEA1, SSEA3, SSEA4, TRA-1-60, and TRA-1-81). Results confirmed these as germline cells with the expression of DAZL (Deleted in Azoospermia-Like) and VASA genes in isolated cells. Immunochemistry results showed that multipotent germline stem cells (mGSCs) expressed these gene markers related to embryonic stem cells (ESCs) and could spontaneously differentiate into three embryonic germ (EG) layers in vitro. The mGSC-derived cardiomyocytes expressed cardiac-specific markers such as sarcomeric alpha actinin, alpha-cardiac actinin; conexin-43, the major protein of gap junctions which are thought to have an important role in the synchronized contraction of the heart and in embryonic development; and cardiac troponin T, the tropomyosin binding subunit of the troponin complex which regulates muscle contraction. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) results indicated that the genes related to cardiac transcription factors were expressed following differentiation. Results of the present study strongly contribute to the information related to the ability of chicken mGSCs to differentiate into cells such as contraction cardiomyocytes similar to ESCs and may provide a new source of cardiomyocytes for basic research and potential therapeutic application in various cardiac degenerative diseases of birds and other animals.

Whole-genome analyses of Korean native and Holstein cattle breeds by massively parallel sequencing.

A main goal of cattle genomics is to identify DNA differences that account for variations in economically important traits. In this study, we performed whole-genome analyses of three important cattle breeds in Korea--Hanwoo, Jeju Heugu, and Korean Holstein--using the Illumina HiSeq 2000 sequencing platform. We achieved 25.5-, 29.6-, and 29.5-fold coverage of the Hanwoo, Jeju Heugu, and Korean Holstein genomes, respectively, and identified a total of 10.4 million single nucleotide polymorphisms (SNPs), of which 54.12% were found to be novel. We also detected 1,063,267 insertions-deletions (InDels) across the genomes (78.92% novel). Annotations of the datasets identified a total of 31,503 nonsynonymous SNPs and 859 frameshift InDels that could affect phenotypic variations in traits of interest. Furthermore, genome-wide copy number variation regions (CNVRs) were detected by comparing the Hanwoo, Jeju Heugu, and previously published Chikso genomes against that of Korean Holstein. A total of 992, 284, and 1881 CNVRs, respectively, were detected throughout the genome. Moreover, 53, 65, 45, and 82 putative regions of homozygosity (ROH) were identified in Hanwoo, Jeju Heugu, Chikso, and Korean Holstein respectively. The results of this study provide a valuable foundation for further investigations to dissect the molecular mechanisms underlying variation in economically important traits in cattle and to develop genetic markers for use in cattle breeding.

Efficacious rat model displays non-toxic effect with Korean beechwood creosote: a possible antibiotic substitute.

Wood creosote, an herbal anti-diarrheal and a mixture of major volatile compounds, was tested for its non-toxicological effects, using a rat model, with the objective to use the creosote as an antibiotic substitute. A total of 30 Sprague-Dawley rats were studied to form five groups with 6 rats each. Korea beechwood creosote was supplemented into three test groups with 0.03 g/kg, 0.07 g/kg and 0.1 g/kg body weight/day without antibiotic support, along with a positive control of Apramycin sulphate (at 0.5% of the daily feed) and a negative control. Korean beechwood creosote supplementation showed no negative effect on the body weight gain in comparison to the negative and the positive control groups and the feed conversion ratio was also comparable with that of the control groups. The clinical pathology parameters studied were also under the umbrella of normal range, including liver specific enzymes, blood glucose, total protein, blood urea nitrogen (BUN), which indicated no toxic effect of creosote at the given doses. The non-hepatotoxic effect was also confirmed using hepatic damage specific molecular markers like Tim-p1, Tim-p2 and Tgf-ß1. The results suggested that Korean beechwood may be used as antibiotic substitute in weanling pigs feed without any toxic effect on the body. Although the antimicrobial properties of creosote were not absolutely similar to those of apramycin sulphate, they were comparable.

Organotypic human spinal cord slice culture as an alternative to direct transplantation of human bone marrow precursor cells for treating spinal cord injury.

OBJECTIVE: To determine the possibility of differentiation of human bone marrow-derived mesenchymal precursor cells (BMDMPCs) into neuronal lineage cells using a human spinal cord organotypic slice coculture technique as an alternative to an in vivo human study. METHODS: Human BMDMPCs were stained with PKH-26 dye before transplantation into 12 human spinal cord slices. In the control group, BMDMPCs were embedded into one spinal cord-free six-well plate containing media. The morphologic differentiation of the transplanted BMDMPCs were observed at 0, 3, 7, and 14 days. Neuroglial differentiation was identified with immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Spherical cells were seen in both groups at day 0. On days 7 and 14, cells developed one or two thick, short processes and typical spindle-shaped cells in the control group and three to five thin, long processes and neuron-like cells in the experimental group. Immunohistochemistry showed double-stained cells with PKH-26 dye (positive) and vimentin (positive), PKH-26 (positive) and neuronal nuclei (NeuN) (positive), and PKH-26 (positive) and glial fibrillary acidic protein (GFAP) (positive) in the experimental group only. RT-PCR showed weak expression of tyrosine kinase A, NeuN, ß-tubulin III, and GFAP in the experimental group. CONCLUSIONS: Organotypic human spinal cord slice culture may be a useful method to verify the neuroglial differentiation of human BMDMPCs as an alternative to a direct human study.

Long-term culture and transplantation of murine testicular germ cells.

The objectives of this study were to develop an in vitro culture system to optimize germ cell proliferation and to measure the potential of the cultured germ cells to produce mature spermatozoa after transplantation into a recipient. Donor germ cells isolated from ROSA26 male mice were cultured with a STO feeder cell layer in Dulbecco's minimal essential medium (DMEM) supplemented with fetal bovine serum (FBS), stem cell factor, leukemia inhibitory factor, basic fibroblast growth factor, insulin-like growth factor 1, interleukin-11, L-glutamine, sodium pyruvate, 2-mercaptoethanol, murine oncostatin M, and platelet-derived growth factor. Donor germ cells formed colonies in the primary cultures after 8-21 days. These cultured colonies were maintained for 4 weeks or longer without subculture and proliferated for up to 8 passages over a period of 3 months. These colonies had alkaline phosphatase activity and incorporated 5-bromo-2'-deoxyuridine. These colonies were positive partially when screened with antibody for germ cell nuclear antigen and c-kit. Germ cells cultured with this supplemented medium showed enhanced colonization vs controls cultured with DMEM and FBS. Cultured germ cells from Rosa26 donors were transplanted into testes and were identified by X-gal staining and histological screening. The cells cultured in the supplemented medium colonized the tubules and initiated spermatogenesis in the recipient mice. This is an improved method for culturing germ cells and may be useful in gene therapy and the production of transgenic animals.