Jae Yong Han

Germ cells and transgenesis in chickens.

Chickens have proven to be useful organisms for transgenic research. This work provides enormous benefits in advancing animal biotechnology and aids in the development of unique technologies for bioreactor production and experimental model development. The various advantages of chicken transgenesis are derived from the genetic and physiological characteristics of this organism, although several physiological properties have impeded the development of an efficient transgenic system. We have developed embryo-mediated and testis-mediated transgenic systems using chicken primordial germ cells (PGCs) from embryos and testicular cells from adult males. These methods are efficient and involve minimal technical effort. Here, we review previous transgenic research using PGCs and testicular cells from chickens. Furthermore, we have summarized the development of the chicken model system in biomedical science and biotechnology and our recent achievements in this field.

Reproduction of Wild Birds via Interspecies Germ Cell Transplantation

Abstract The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5%. In conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species..

Cloning and functional characterization of chicken interleukin-17D

The chicken interleukin-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 13.3kDa. Comparison of the deduced amino acid sequence of chIL-17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity, respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL-17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL-17D mRNA were up-regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL-17D mRNA in CD4(+), CD8(+) and TCR1(+) intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2(+) cells. Treatment of CHCC-OU2 fibroblasts with chIL-17D recombinant protein induced the expression of IL-6 and IL-8. Collectively, these results suggest that chL-17D has structural and functional similarities to mammalian IL-17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.

Conserved expression pattern of chicken DAZL in primordial germ cells and germ-line cells

The autosomal gene deleted in azoospermia-like (DAZL), which was identified as a member of the deleted in azoospermia (DAZ) family, is homologous to the Drosophila gene BOULE. The authors investigated the sequence similarities of chicken DAZL (cDAZL) with several invertebrate and vertebrate DAZL proteins using CLUSTAL X. A comparison of the primary sequence of cDAZL with other DAZL proteins indicated significant similarities: 70-82% with reptiles, 63-68% with mammals, 51-67% with amphibians, and 42-49% with fishes. The conserved expression pattern of cDAZL was examined by reverse transcription-PCR, quantitative real-time PCR, and in situ hybridization during primordial germ cell (PGC) settlement in the gonads and germ-line development. Among several tissues examined on embryonic day E6.5, DAZL expression was detected specifically in male and female gonads. Quantitative real-time PCR and in situ hybridization revealed strong cDAZL expression in PGCs. When the PGCs differentiated into germ cells, cDAZL expression was slightly decreased; however, expression was continuously detected in germ-line cells until the adult stage. We inferred that cDAZL expression was conserved in PGCs and during germ-line differentiation until the adult stage, making them a valuable molecular marker for studies of PGC differentiation and germ-line development in chickens.

The study on production and performance of crossbred Korean native chickens (KNC).

Department of Agricultural Biotechnology, Seoul National University, Seoul, KoreaABSTRACT The current work was carried out to investigate the effect of crossbred Korean native chickens (KNC) on performance and carcass ratio. Seven hundred twenty 1-d-old chicks were divided into groups by strain (A, B, C and D) and sex (male and female). Strains were A) (KNC egg-meat type C strains×KNC meat type S strains)×KNC meat type H strains, B) (KNC egg-meat type C strains×KNC meat type H strains)×KNC meat type S strains, C) (KNC native R strains×KNC meat type S strains)×KNC meat type H strains and D) (KNC native L strains×KNC meat type H strains×Ross broiler. Experimental diets consisted of 3 phases such as starter(05 weeks; CP 20.0%, ME 3,050 kcal/kg), earlier (58 weeks; CP 18.0%, ME 3,100 kcal/kg) and finisher (812 weeks; CP 16.0%, ME 3,150 kcal/kg). Body weight (BW) and feed intake (FI) was measured every week and carcass ratio(CR) was calculated at 5 and 10 week after starting experiment. There was no difference in BW among strains until 5 weeks (P>0.05), however D strain resulted in a higher BW after 5 weeks (P<0.05). Body weight gain (BWG) and FI in D strain were also significant higher compared to the other strains for all periods. However, D strains showed the lowest (P<0.05) fee conversion ratio (FCR). The other strains except D showed a similar BW, BWG, FI and FCR among strains. In addition, there were no differences in carcass weight (CW) and carcass ratio (CR) among strains at 5 weeks, however D strain showed higher CW and CR at 10 weeks. These results suggested the basic data that needed to develope the new strains.(Key words : Korean native chickens, crossbred, performance, carcass ratio)

Genetic modification of chicken germ cells.

Over the past two decades numerous reports have demonstrated that the genetic modification of poultry genomes has great potential for improving poultry production; moreover, it may be used as a powerful tool for the production of industrial proteins. To date, transgenic techniques have been established for generating transgenic birds that express recombinant human proteins in hen eggs, as well as tissue‐specific genes as an animal model. The production of transgenic birds is a promising approach that could have practical applications in agriculture and biopharmacology, in addition to advancing our understanding of avian biology. Finally, germ cell–mediated transgenesis could provide a more efficient strategy for creating gene‐targeted insertions and deletions in avian species.

Chemical Composition and Meat Quality of Crossbred Korean Native Chickens (KNC)

This work was carried out to investigate chemical composition and meat quality of crossbred Korean native chickens (KNC). Ninety 1-d male chicks were used in this work and were divided into 4 groups as A: (KNC egg-meat type C strains × KNC meat type S strains) ( ) × KNC meat type H strains ( ), B: (KNC egg-meat type C strains × KNC meat type H strains) ( ) × KNC meat type S strains ( ), C: (KNC native R strains × KNC meat type S strains) ( ) × KNC meat type H strains ( ), D: (KNC native L strains × KNC meat type H strains) ( ) × Ross broiler ( ) strains. They were fed the broiler diets for 12 weeks at the flat house and twenty seven chickens were slaughtered at week 5 and 10. Chicken thigh and breast were weighed and physicochemical compositions and sensory characteristics were investigated. Physical compositions of meats have no difference among strains at 5 week, and B strains differed from other strains at 10 week (P<0.05). The pH, moisture, and collagen content of meat from B strains were lower than other strains at 5 week. Ash and collagen of A strains were the lowest at 10 week (P<0.05), but others have no difference among strains. In sensory test, juiciness of D strains was the highest at 5 week, and tenderness of B strains was the lowest at 10 week (P<0.05). In conclusion, the crossbreeding of KNC did not affect physical traits but affected chemical composition of the chicken meat slaughtered at 5 week.

Genetic Variations of Chicken MC1R Gene and Associations with Feather Color of Korean Native Chicken (KNC) `Woorimatdag`

Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, KoreaABSTRACT There are several loci controlling the feather color of birds, of which one of the most studied is Extended black (E) encoding the melanocortin 1-receptor (MC1R). Mutations in this gene affect the relative distribution of eumelanin, phaeomelanin. The association of feather color and sequence polymorphism in the melanocortin 1-receptor (MC1R) gene was investigated using Korean native chicken H breed (H_PL) and ‘Woorimatdag’ commercial chickens (Woorimatdag_CC). In order to correlate gene mutation to Korean native chicken feather color, single nucleotide polymorphism (SNP) from MC1R gene sequence were investigated. A total of 307 birds from H_PL and Woorimatdag_CC were used. H_PL have black, black-brown feather color and Woorimatdag_CC have black with brown spots or brown with black spots. There are 6 SNPs in MC1R gene, locus T69C, C212T, A274G, G376A, G636A, T637C. 3 SNPs are nonsynonymous that change amino acid. But it is difficult to find correlation of feather color and polymorphisms. It will be needed to increase the population of Korean native chicken H breed and correlation analysis of genetic variation with feather colors.(Key words : Korean Native Chickens, feather color, MC1R, SNP)

Genotoxicity Assessment of HM10620 Containing Recombinant Human Interferon-α

HM10620 is a recombinant human interferon-α (rhIFN-α) linked to immunoglobulin via N-terminal–specific non-peptidyl polyethylene glycol linker to improve the in vivo stability of interferon. Potential genotoxic effects of HM10620 in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro chromosomal aberration assay, and the in vivo micronucleus assay. HM10620 did not cause any mutation in the Ames assay tested using five tester strains at six concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg/plate. To assess clastogenic effect, the in vitro chromosomal aberration assay and the in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Chromosomal aberration was not induced at the concentrations of 10, 20, and 40 μg/mL. Also, there was no difference in the incidence of micronucleated polychromatic erythrocytes at doses of 10, 20, and 40 mg/kg in male mice compared with the vehicle control group. Therefore, based on the results obtained from the three studies, it is concluded that HM10620 is not a mutagenic agent in bacterial cells and causes no chromosomal damage in mammalian cells both in vitro and in vivo.

Gene pathways and cell cycle-related genes in cultured avian primordial germ cells

Primordial germ cells (PGC) from early embryos are applicable to various kinds of research, including the production of transgenic animals. Primordial germ cells eventually migrate and differentiate into germ cells in the gonads, where they settle and rapidly proliferate. However, the proliferation rate of PGC is low in early embryos, and there are many significant pathways that mediate PGC activity. Therefore, in vitro culture of PGC from early embryos with efficient growth factors has been necessary. Recently, we cultured chicken PGC from embryonic d 2.5 with basic fibroblast growth factor and characterized the PGC through analysis of cell morphology, survival, proliferation, and apoptosis. However, large-scale analyses of genes expressed in cultured PGC and the genes involved in associated pathways are limited. The objective of the present investigation was to identify the signaling and metabolic pathways of expressed genes by microarray comparison between PGC and their somatic counterpart, chicken embryonic fibroblasts (CEF). We identified 795 genes that were expressed more predominantly in PGC and 824 genes that were expressed more predominantly in CEF. Among the predominant genes in PGC, 201 were differentially identified in 106 pathways. Among the predominant genes in CEF, 242 were differentially identified in 99 pathways. To further validate the genes involved in at least one candidate pathway, those involved in the cell cycle (12 predominant genes in PGC and 8 predominant genes in CEF) were examined by real-time PCR. To the best of our knowledge, this study is the first to investigate signaling and metabolic pathways in cultured PGC.