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

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

Malignant Fibrous Histiocytoma with In-Transit Metastasis

Malignant fibrous histiocytoma (MFH) is the most common fibroblastic tumor, but its cutaneous metastasis, especially in-transit metastasis, is extremely rare. We describe the case of a 30-year-old Japanese man with a recurrent MFH on the scalp accompanied by in-transit metastasis, which had been treated as a benign skin tumor 8 years before. The main bulk of the recurrent tumor was located in the dermis, but the metastatic tumor was mainly located in the subcutis. Generally, atypical fibroxanthoma, also known as cutaneous MFH, is rarely metastasized and presents a benign clinical course. Since there is a great difference between the prognosis of MFH and atypical fibroxanthoma, precise diagnosis of the primary tumor is essential

Culture Conditions for Maintain Propagation, Long-term Survival and Germline Transmission of Chicken Primordial Germ Cell-Like Cells

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.ArticleJOURNAL OF POULTRY SCIENCE. 51(1):87-95 (2014)journal articl

Depletion of Primordial Germ Cells (PGCs) by X-irradiation to Extraembryonic Region of Chicken Embryos and Expression of Xenotransplanted Quail PGCs

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12-3 x 0.25 mm, similar to 0.1 g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (similar to 7.2 Gy) at stages 13 to 17 showed a hatchability of 35% (106/301). whereas the hatchability of unshielded embryos was 26% (27/105). The relative Population of gonadal PGCs Lit stage 30 for embryos irradiated at stage 13 with or Without shielding was 13 and 5%. respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or Without shielding and transfused with quail embryonic blood containing PGCs each exhibited similar to 130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to similar to 7.2 Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.ArticleJOURNAL OF POULTRY SCIENCE. 46(2): 136-143(2009)journal articl

Genome-wide investigation of in vivo EGR-1 binding sites in monocytic differentiation

A Genome-wide analysis of EGR-1 binding sites reveals co-localization with CpG islands and histone H3 lysine 9 binding. SP-1 binding occupancies near EGR-1 binding sites are dramatically altered

Rb Regulates DNA Damage Response and Cellular Senescence through E2F-Dependent Suppression of N-Ras Isoprenylation

Oncogene-induced cellular senescence is well documented, but little is known about how infinite cell proliferation induced by loss of tumor suppressor genes is antagonized by cellular functions. Rb heterozygous mice generate Rb-deficient C cell adenomas that progress to adenocarcinomas following biallelic loss of N-ras. Here, we demonstrate that pRb inactivation induces aberrant expression of farnesyl diphosphate synthase, many prenyltransferases, and their upstream regulators sterol regulatory element-binding proteins (SREBPs) in an E2F-dependent manner, leading to enhanced isoprenylation and activation of N-Ras. Consequently, elevated N-Ras activity induces DNA damage response and p130-dependent cellular senescence in Rb-deficient cells. Furthermore, Rb heterozygous mice additionally lacking any of Ink4a, Arf, or Suv39h1 generated C cell adenocarcinomas, suggesting that cellular senescence antagonizes Rb-deficient carcinogenesis. © 2009 Elsevier Inc. All rights reserved

Comparison of Dielectric Properties between Epoxy Composites with Nanosized Clay Fillers Modified by Primary Amine and Tertiary Amine (Final draft (Post-print) version)

Epoxy-based nanocomposites (NCs) were prepared using clay modified by either primary amine or tertiary amine, and the effect of the difference in modifier on the thermal and dielectric properties of the NCs were discussed. The NC with clay fillers modified by the primary amine, 1C, shows a glass transition end temperature (Teg) at a temperature 20°C lower than the neat epoxy (N). This indicates that the resin of 1C is less crosslinked than that of N. On the other hand, the sample 3C, in which the clay was modified by the tertiary amine, shows a DSC spectrum close to that of N. Namely, 3C has a high crosslinking density similar to N. While the three samples show a relaxation peak in their dielectric loss spectra, the peak appears at high frequencies in 1C compared to N and 3C. Moreover, ionic conduction current flows more at high temperatures in 1C than in N or 3C. These facts are ascribable to the difference in their crosslinking densities

Dielectric properties of epoxy/clay nanocomposites : effects of curing agent and clay dispersion method (Final draft (Post-print) version)

Effects of the differences in the curing agent and filler dispersion method on the dielectric properties were examined for epoxy/clay nanocomposites. Irrespective of the clay dispersion method, relative permittivity and electrical conductivity are higher in the samples cured with the amine. Moreover, negative heterocharge accumulates in the vicinity of the anode in the amine-cured samples, whereas positive homocharge accumulates in the acid anhydride-cured samples. From the results of UV photon absorption and PL measurement, the bandgap or the energy at which the photon absorption increases drastically is smaller in the amine-cured samples than in the acid anhydride-cured samples. Ion migration can occur easily in the amine-cured samples whose electrical conductivity and relative permittivity are higher than the acid anhydride-cured samples. The curing agent gives the strongest effect, while the existence of clay affects secondly and the filler dispersion method has the weakest effect