Regeneration of Muscular Dystrophy Chickens by Transplantation of Early Blastodermal Cells into Recipient Embryos

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy. It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens.ArticleJOURNAL OF POULTRY SCIENCE. 46(1): 46-51(2009)journal articl

Transcription of Endogenous Retrovirus Group K Members and Their Neighboring Genes in Chicken Skeletal Muscle Myoblasts

Skeletal muscle myoblasts are myogenic precursor cells that generate myofibers during muscle development and growth. We recently reported that broiler myoblasts, compared to layer myoblasts, proliferate and differentiate more actively and promptly into myocytes, which corresponds well with the muscle phenotype of broilers. Furthermore, RNA sequencing (RNA-seq) revealed that numerous genes are differentially expressed between layer and broiler myoblasts during myogenic differentiation. Based on the RNA-seq data, we herein report that chicken myoblasts transcribe endogenous retrovirus group K member (ERVK) genes. In total, 16 ERVKs were highly expressed in layer myoblasts and two (termed BrK1 and BrK2) were significantly induced in broiler myoblasts. These transcribed ERVKs had a totalof 182 neighboring genes within ±100 kb on the chromosomes, of which 40% were concentrated within ±10 kb of the ERVKs. We further investigated whether the transcription of ERVKs affects the expression of their neighboring genes. BrK1 had two neighboring genes; LOC107052719 was overlapping with BrK1 and downregulated in the broiler myoblasts, and FAM19A2 was upregulated in the broiler myoblasts as well as BrK1. BrK2 had 14 neighboring genes, and only one gene, LOC772243, was differentially expressed between layer and broiler myoblasts. LOC772243 was overlapping with BrK2 and suppressed in the broiler myoblasts. These data indicate that the transcription of ERVKs may impact the expression of their neighboring genes in chicken myoblasts.ArticleThe Journal of Poultry Science. 58(2) :79-87(2021)journal articl

Restriction of Germline Proliferation by Soft X-ray Irradiation of Chicken Embryos and its Application to Chimera Production

Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the efficiency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for 0, 20, 40 or 60s before incubation. The irradiated embryos manifested delayed development at 60 h of incubation. They also showed reduced numbers of circulating PGCs at stages 14 and 15 and of gonadal PGCs at stage 30. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for 20 s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for 20 s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage 30. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the efficiency of incorporation of donor PGCs.ArticleJOURNAL OF POULTRY SCIENCE. 45(4): 292-297(2008)journal articl

Toll -like receptor ligand-dependent inflammatory responses in chick skeletal muscle myoblasts

Epub 2018 Oct 30Toll-like receptors (TLRs) are a group of sensory receptors which are capable of recognizing a microbial invasion and activating innate immune system responses, including inflammatory responses, in both immune and non immune cells. However, TLR functions in chick myoblasts, which are myogenic precursor cells contributing to skeletal muscle development and growth, have not been studied. Here, we report the expression patterns of TLR genes as well as TLR ligand-dependent transcriptions of interleukin (IL) genes in primary-cultured chick myoblasts. Almost TLR genes were expressed both in layer and broiler myoblasts but TLR1A was detected only in embryonic layer chick myoblasts. Chick TLR1/2 ligands, Pam(3)CSK(4) and FSL-1, induced inflammatory ILs in both layer and broiler myoblasts but a TLR4 ligand, lipopolysaccharide, scarcely promoted. This is the first report on TLR ligand-dependent inflammatory responses in chick myoblasts, which may provide useful information to chicken breeding and meat production industries.ArticleDEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY. 91:115-122 (2019)journal articl

Complete Regeneration of Muscular Dystrophy Chickens by Mating of Male and Female Offspring Derived from Germline Chimeras

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated. In the present studies, sexually matured males and females of the type-l were mated each other. Form these mating. chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of Muscular dystrophy. were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens Could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and Survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain. The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy.ArticleJOURNAL OF POULTRY SCIENCE. 46(2): 123-126(2009)journal articl

Simple Culture System for Bobwhite Quail and Japanese Quail Embryos from the Blastoderm Stage to Hatching using a Single Surrogate Eggshell

The ex vivo culture of avian embryos is a technique for the long-term culturing of embryos outside of their own shell and shell membrane. It allows easy access to the developing embryos and embryo manipulation. The two-step system is widely applied when the culture is performed after oviposition. Japanese quail as well as bobwhite quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been more limited than those on the Japanese quail. We have developed a more simplified ex vivo culture protocol for the two species of quail embryos from the blastoderm stage through to hatching using a single surrogate eggshell. Hatchabilities of 31% and 27% were obtained in bobwhite quail and Japanese quail embryos, respectively. The simple system described in the present study is an easy and acceptable procedure.ArticleJOURNAL OF POULTRY SCIENCE. 51(2):202-205 (2014)journal articl

Autonomous xenogenic cell fusion of murine and chick skeletal muscle myoblasts

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP(+)/DsRed(+) hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products.ArticleANIMAL SCIENCE JOURNAL. 88(11):1880-1885 (2017)journal articl

Analysis of Developmental Changes in Avian DNA Methylation Using a Novel Method for Quantifying Genome-wide DNA Methylation

Individual differentiated somatic cells and undifferentiated stem cells have common genome, although their functions or morphological characters are very different. These differences are derived from difference of gene expression pattern. DNA methylation is generally key factor of Suppression of gene and its level is globally change during mammalian early development. But, in birds, whether genome-wide changes in DNA methylation occur during embryonic development is still unknown. Here, we show that genome-wide DNA methylation to assess occurrence during early chick embryonic development. We found that the methylation status at stage 1 was approximately 57%, after which it gradually decreases, reaching a minimum at stage 10 (33%). After stage 10, DNA methylation gradually increased. These results should contribute to clarify the epigenetic mechanisms in birds.ArticleJOURNAL OF POULTRY SCIENCE. 46(4): 286-290(2009)journal articl

Culture System for Bobwhite Quail Embryos from the Blastoderm Stage to Hatching

Quail are divided phylogenetically into two groups, Old World quail and New World quail. Old World quail, such as the Japanese quail (Coturnix japonica), belong to the Phasianidae and distributed in the Palaearctic region (Europe, North Africa, and Asia), whereas New World quail, such as the bobwhite quail (Colinus virginianus), belong to the Odontophoridae and are restricted to North and South America. Both the bobwhite quail and the Japanese quail are used as models for avian safety assessment as recommended by the Organisation for Economic Co-operation and Development (OECD) guidelines. However, biological studies on the bobwhite quail have been limited compared with those on the Japanese quail. We have therefore now developed an ex vivo culture protocol for bobwhite quail embryos from the blastoderm stage through hatching. Of the various culture conditions examined in the present study, a good hatching rate (39%) was obtained when the embryos were cultured ex vivo in a two-step procedure. Unincubated embryos (with egg yolk) were first cultured inside the shell of a Japanese quail egg (11.5 to 13.0 g whole egg weight) together with chicken thin albumen for 63 to 65 h and were then transferred to the shell of a small-sized chicken egg (38 g whole egg weight) until hatching. This ex vivo culture system should provide to be widely applicable to the maintenance and generation of manipulated birds for basic and applied studies on the bobwhite quail.ArticleJOURNAL OF POULTRY SCIENCE. 50(2):155-158 (2013)journal articl

A Novel Concentrating System of Chicken Stem Cells by Bone Marrow Side Population Cells

Numerous studies in mammalian species have recently been reported that many stem cells have an ability to efficiently efflux the vital DNA-binding dye Hoechst 33342, and it is called side population (SP) cells. However, few study have been reported on the avian SP cells. It could be possible that concentration of hematopoietic stem cells (HSCs) in birds since the characteristic of SIP cells should be shared in various tissues and species. In this study, we first attempted the isolation of SP cells from chicken bone marrow and the assessment by gene expression and morphologic analyses. Bone marrow cells (BMCs) were flushed from the femurs and tibias of chicks aged at 10 days with PBS. The BMCs were layered on lymphocyte separation medium and centrifuged for excluding the erythrocytes. The separated cells were adjusted to 10(6)/ml in HBSS. Hoechst 33342 were added (1.25 mu g/ml) and incubated 60 to 90 minutes at 37 degrees C. Propidium iodide was added (2 mu g/ml) to exclude dead cells. The SP cells were isolated with flow cytometer. The sorted cells were stained with May-Gruenwald Giemsa (MG) for morphological analysis and RNA was extracted for gene expression analysis. The avian SP cells which was vanished by addition verapamil counld be separated. The percentage of SP cells in chicken bone marrow was about 2.6%. The morphological analysis by MG staining indicated that the SP cells had a larger nuclear and little cytoplasm which were typical characterisation of mouse HSCs. The pattern of gene expressions (CD34, c-Kit, CD4 and CD8) in SP cells also resembled that of the mouse HSCs. These results suggested that the HSCs could be enriched from avian bone marrow cells. Together with these results, it was concluded that SP is one of powerful tools for concentration of avian stem cells.ArticleJOURNAL OF POULTRY SCIENCE. 47(1): 53-56(2010)journal articl