• Asian-Australasian Journal of Animal Sciences
• /
• v.6 no.4
• /
• pp.581-589
• /
• 1993

A plasmid vector, $RSVLTR/{\beta}G2$, containing lacZ gene under the control of the RSVLTR promoter were transfected into chicken embryo fibroblasts by three different transfection methods. Calcium phosphate, lipsome and DEAE-dextran techniques were applied for transfection of chicken cells. A histochemical assay with X-gal was used as a simple method for screening transfected cells. Plasmid $RSVLTR/{\beta}G2$ was expressed proficiently in the chicken embryo fibroblast. Calcium phosphate-DNA precipitate transfection resulted in the highest efficiency for transient expression of $RSVLTR/{\beta}G2$. Transfected cells formed colonies on the 9th day of incubation indicating stable transformation of the inserted plasmid.

• Korean Journal of Veterinary Research
• /
• v.31 no.4
• /
• pp.471-477
• /
• 1991

Attempts to isolate chicken anemia agent (CAA) were made by inoculating tissue homogenates into MDCC-MSBl or LSCC-1104B1 cell lines and passaging the cells serially. CAA was isolated from the liver and thymus of 11 weeks old layer chickens and from the liver of 10 weeks old broiler breeder chickens. The layer flock experienced approximately 45% mortality during 9 to 14 week of age from gangrenous dermatitis and lymphoid organs of affected chickens were severely atrophied. The broiler breeder flock experienced approximately 7% mortality during 7 to 9 weeks of age and affected birds showed lesions of colibacillosis, staphylococcal arthritis, and coccidiosis together with atrophied lymphoid organs. The isolated viruses were identified as CAA by the indirect fluorescent antibody test and virus neutralization test using CAA immune sera including one to Gifu-1 strain of CAA. The CAA isolate 89-69, when inoculated into susceptible 1 day old SPF chicks, induced anemia 14 to 16 days after inoculation. It did not induce any cytopathic effects in chicken embryo liver and chicken embryo fibroblast cell cultures. Infectivity of the isolate was not affected by the treatment of chloroform or heat ($70^{\circ}C$ for 15 minutes).

• Animal Bioscience
• /
• v.36 no.4
• /
• pp.570-583
• /
• 2023

Objective: Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. Methods: Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. Results: A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen-activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R² = 0.92-0.95, p<0.01). Conclusion: This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

• Asian-Australasian Journal of Animal Sciences
• /
• v.9 no.6
• /
• pp.701-703
• /
• 1996

The influence of phenylalanine (Phe) in the medium on protein synthesis of chicken embryo fibroblasts (CEF) was examined. CEF was derived from 9-d-old embryos by trypsin-EDTA digestion. To examine the deficiency of Phe in the medium, CEF was cultured in Dulbecco's modified Eagle's medium (DMEM) with or without Phe. CEF was also cultured in Dulbecco's phosphate buffered saline (PBS ($Ca^{2+}$, $Mg^{2+}$)) with or without $400{\mu}m$ Phe in order to examine the effect of Phe supplementation. All media were supplemented with 10% (v/v) fetal calf serum. After incubation for 6, 30 and 54 h, protein synthesis was measured by the incorporation of L-[2, $6-^{3}H$] Phe into CEF for further 18 h. Protein synthesis of CEF cultured in DMEM was higher than that in PBS ($Ca^{2+}$, $Mg^{2+}$). High specific radioactivity of Phe due to the low concentration of Phe in the medium resulted in the apparent increase in protein synthesis of CEF. Protein synthesis cultured in PBS ($Ca^{2+}$, $Mg^{2+}$) with Phe did not increase during 72 h of cell culture.

• Korean Journal of Poultry Science
• /
• v.27 no.1
• /
• pp.73-78
• /
• 2000

Embryonic stem (ES) cells are pluripotent cell lines, which derived from preimplantation embryo. These cells have been used as a vehicle of foreign DNA for production of transgenic mammals. this experiment was performed to examined the possible use of blastodermal cells derived from hen's egg for germline manipulation. Stage X blsdtodermal cells isolated from fertilized eggs were cultured in DMEM containing 15% fetal calf serum. Blastodermal cells wre co-cultured on the chicken embryonic fibroblast (CEF) or mouse embryonic fibroblast(MEF) cells. to examine the effects of growth factors on stem cell growth, bFGF and LIF were added. There was no significant difference in colony formation of putative ES cells between CEF and MEF as a feederlayer, but the addition of growth factors enhanced the proliferation and inhibited differentiation of blastodermal cells. To characterize the cell colonies as a putative ES cells, putative embryonic cell colonies were stained by periodic acid Schiffs (PAS) reagent. The putative ES cell colonies showed intensive positive reaction similar to the property of undifferentiated PGC upto 20days in vitro, but not in other cell types. this result demonstrates that PAS-positive cell colonies may be used for the study of establishment of chicken ES cell lines for the production of transgenic chicken.

• Animal Bioscience
• /
• v.36 no.7
• /
• pp.1120-1129
• /
• 2023

Objective: This study aimed to determine the protective efficacy of Buddha's Temple (BT) extract against tert-butyl hydroperoxide (t-BHP)-induced oxidative stress in Gallus gallus chicken embryo fibroblast cell line (DF-1) and its effects on the cell lipid metabolism. Methods: In this experimental study, Gallus gallus DF-1 fibroblast cells were pretreated with BT 10^-7 for 24 hours, followed by their six-hour exposure to t-BHP (100 μM). Water-soluble tetrazolium salt-8 (WST-8) assays were performed, and the growth curve was computed. The intracellular gene expression changes caused by BT extract were confirmed through quantitative polymerase chain reaction (qPCR). Flow cytometry, oil red O staining experiment, and thin-layer chromatography were performed for the detection of intracellular metabolic mechanism changes. Results: The WST-8 assay results showed that the BT pretreatment of Gallus gallus DF-1 fibroblast cell increased their cell survival rate by 1.08%±0.04%, decreased the reactive oxygen species (ROS) level by 0.93%±0.12% even after exposure to oxidants, and stabilized mitochondrial activity by 1.37%±0.36%. In addition, qPCR results confirmed that the gene expression levels of tumor necrosis factor α (TNFα), TIR domain-containing adapter inducing IFN-beta (TICAM1), and glucose-regulated protein 78 (GRP78) were regulated, which contributed to cell stabilization. Thin-layer chromatography and oil red O analyses showed a clear decrease in the contents of lipid metabolites such as triacylglycerol and free fatty acids. Conclusion: In this study, we confirmed that the examined BT extract exerted selective protective effects on Gallus gallus DF-1 fibroblast cells against cell damage caused by t-BHP, which is a strong oxidative inducer. Furthermore, we established that this extract significantly reduced the intracellular ROS accumulation due to oxidative stress, which contributes to an increase in poultry production and higher incomes.

• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2001.11a
• /
• pp.40-46
• /
• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Korean Journal of Poultry Science
• /
• v.12 no.2
• /
• pp.135-143
• /
• 1985

A total of eight strains of avian reoviruses were isolated from chickens with arthritis or stunted growth. The isolations were made from broilers or broiler breeders under 12 weeks of age. The viruses had a typical morphology of reoviruses with double capsid layers and 81nm of diameter. In agar gel precipitation tests, the isolates reacted with antisera prepared against S-1133 or R-1 strains of avian reoviruses and cross reacted with S-1133 antigen. They did not agglutinated RBC's from day-old chicks, adult chickens, guinea pigs, and horses. The isolates showed strong resistance against the treatments of chloroform, IUdR, and heat, When infectivities of the viruses were titrated in cell cultures of chicken embryo fibroblast, chicken embryo liver, and Vero cells, similar end points reached four to five days after inoculation, regardless of tell types and virus inoculation time, either inoculated simultaneously at the time of cell seeding or on confluency. Mean times of mortality of chicken embryos inoculated with the isolates via the chorioallantoic membrane ranged from 54 to 59 hours and that of S-1133 strain was 73 hours.

• Korean Journal of Veterinary Research
• /
• v.45 no.4
• /
• pp.569-574
• /
• 2005

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to reticuloendotheliosis virus (REV) at single serum dilution was standardized. REV HI, one of the Korean field isolates, was inoculated into chicken embryo fibroblast (CEF) cells and was harvested from the culture fluids and cells after 10 to 12 days. Viruses were purified by centrifugation at the $107,000{\times}g$ for 12 hours on 20, 30, 45% (W/V) sucrose gradient. Virus specific fraction was collected and used as ELISA antigen. To standardize ELISA, the optimal concentration of coating antigen ($1{\mu}g/well$) and conjugate (1/1000) was determined by corrected OD (OD value of positive serum-OD value of negative serum) and P/N ratio (OD value of positive serum/OD value of negative serum). To calculate ELISA titer by measuring absorbance at 1/400 single serum dilution, serum titrations were carried out for various sample sera together with standard positive and negative sera. The observed titers of serum samples were plotted against sample/positive (s/p) ratios at 1/400 serum dilution. From the above data, the ELISA titers could be calculated by the equation of $log_{10}$ ELISA titer = 2.2763 ($log_{10}$ s/p) + 3.482 (r = 0.93). For evaluating the sensitivity, the standardized method were compared with conventional agar gel immunodiffusion (AGID) test method using serum samples collected from REV infected field chicken flocks. Fifty seven of 60 samples (95%) were positive for REV by ELISA, whereas only 11 (18.3%) samples were positive by AGID test. This results suggested that the ELISA tests developed in this study could be used for detection of antibodies to REV with high sensitivity.

• Korean Journal of Poultry Science
• /
• v.27 no.2
• /
• pp.109-114
• /
• 2000

The objective of these experiments was to develop an attenuated thermostable Newcastle disease virus(NDV), CBP-1 strain isolated from infected pheasants. Safety, pathogenicity and protective effects against velogenic NDV were also investigated to evaluate if the attenuated NDV, CBP-1 strain could be a candidate for a new NDV vaccine strain. CBP-1 strain was passaged up to the 173 times by nine days old embryonated eggs and chicken embryo fibroblast(CEF) cell cultures. Its sensitivitly to lipid solvents and low pH, thermostability, mean death time(MDT), intracerebral pathogenicity index(ICPI) of one day old chicks and intravenous pathogenicity index(IVPI) of four weeks old chicks were examined. Safety, boosting and protective effects were tested by chicks mortality. CBP-1 NDV strain had significant thermostability at 56$\^{C}$ for 30 minutes. by hemagglutinin activity and egg infectivity test, but was not resistant to lipid solvent. It showed possibility to use as a feed or water vaccine because of the resistance to low pH. MDT, ICPI and IVPI of CBP-1 were attenuated from 51.5, 1.96, 2.60 to 112.4, 1.12, 1.45. These results implied that the 173rd passages in embryonated egg and CEF cell cultures induced a substantial attenuation of the pathogenicity of the parent virus, changing the virulence from velogenic to intermediate between mesogenic and lentogenic. After vaccination with CBP-1 at one day old by drinking water mortality was 17.5%. However, spray vaccination with B1 at one day old, CBP-1 at two weeks ild and challenge with velogenic Kyojeongwon strain at four weeks old showed 93.5% survival rate. Mortality of chicks, vaccination with 173rd passaged CBP-1 strain at one day old, two weeks old and challenge with Kyokeongwon strain at four weeks old, was 20.0%. The results of these studies indicated that partial attenuated CBP-1 strain tended to be a low safety for ND of broiler chicks and would need to be more successive attenuation.