Perspectives on avian stem cells for poultry breeding

Stem cells have prulipotency to differentiate into many types of cell lineages. Recent progress of avian biotechnology enabled us to analyze the developmental fate of the stem cells: embryonic stem cells / primordial germ cells (PGCs). The stem cells were identified in the central area of the area pellucida of the stage X blastoderms. These cells could be applied for production of germline chimeras and organ regeneration. Generation of medical substrate in transgenic chickens has considerable interests in pharmaceuticals. Sex alteration of the offspring should be enormously beneficial to the poultry industry. Fertilization of the sex-reversed sperm could lead to sexual alteration of the offspring. These strategies using stem cells / PGCs should be one of the most powerful tools for future poultry breeding.ArticleANIMAL SCIENCE JOURNAL.87(9):1065-1075(2016)journal articl

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

Isotopic Analysis of Rb and Sr Using a Full Automatic Thermal Ionization Mass Spectrometer

Analytical method for strontium isotope ratios and rubidium and strontium concentrations has been established using a full automatic thermal ionization mass spectrometer. The machine is a modified model "MAT 260" of Varian MAT LTD. Each of simultaneously loaded thirteen samples is successively analysed full automatically following to a specific controling program which has been selected as being most suitable for each sample. However, the most characteristic feature of this machine compared to other types is computer controled peak jumping and peak centering before measurment of the signal of each peak. By this the accuracy of isotopic measurement has been surprisingly improved. Analytical procedures are described in detail which include decomposition of samples, separation of Rb and Sr, loading samples on filament and mass spectrometry. Accuracy and reproducibility of isotope analyses are excellent : 43 separate analyses of standard sample NBS 987 over one year gave a mean (87)Sr/(86)Sr ratio of 0.710238 (normalized to the (88)Sr/(86)Sr ratio of 8.375209) with a value of　20δ of 0.000008. The ratio obtained is slightly　higher than the value of 0.71014 given by NBS,　but it is almost identical to the mean of recently　reported twenty analyses. Our determinations for Rb and Sr concentrations of standard samples are as follows : JG-1, Rb 184.4 ppm, Sr 185.5 ppm; JB-l, Rb 41.5Ppm, Sr 448.4 ppm , each of which is near the mean of reported values for corresponding element of the sample (ANDO et al., 1974). Concentrations of Rb and Sr in pure water and regents used in chemical treatement of samples were also measured; a possible error due to the contamination is negligible for most geochemical samples

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

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

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

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

Targeted mutagenesis in chicken using CRISPR/Cas9 system

The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (> 90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens.ArticleSCIENTIFIC REPORTS.6:23980(2016)journal articl