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Developmental Genetic Analysis of Avian Primordial Germ Cells and the Application to Poultry Biotechnology

  • Kagami, H.
    • Korean Journal of Poultry Science
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    • v.28 no.2
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    • pp.135-142
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    • 2001
  • A novel sterategy has been established to determine the origin of the Primordial Germ Cells (PGCs) in avian embryos directly and the developmental fate of the PGCs for the application to Poultry biotechnology. Cells were removed from 1) the centre of area pellucida, 2) the outer of area pellucida and 3) the area opaca of the stage X blastoderm (Eyal-Giladi & Kochav, 1976). When the cells were removed from the centre of area pellucida, the mean number of circulating PGCs in blood was significantly decreased in the embryo at stage 15 (Hamburger & Hamilton, 1951) as compared to intact embryos. When the cells were replenished with donor cells, no reduction in the PGCs number was observed. The removal of cells at the outer of area pellucida or at the area opaca had no effect on the number of PGCs. In case, another set of the manipulated embryos were cultured ex vivo to the hatching and reared to the sexual maturity, the absence of germ cells and degeneration of seminiferous tubules was observed in resulting chickens derived from the blastoderm in which the cells were removed from the centre of the area pellucida. It was concluded that the avian Primordial Germ cells are originated at the center of area pellucida. Developmental ability of the cells to differentiate into somatic cells and germ cells in chimeras were analyzed. Somatic chimerism was detected as black feather attributed from donor cells. Molecular identification by use of female - specific DNA was performed. It was confirmed that the donor cells could be differentiated into chimeric body and erythrocytes. Donor cells retained the ability to differentiate into germline in chimeric gonads. More than 70% of the generated chimeras transmitted donor derived gametes to their offspring indicating that the cells at the center of area pellucida had the high ability to differentiate into germ cells. A molecular technique to identify germline chimerism has been developed by use of gene scan analysis. Strain specific DNA fragments were amplified by the method. It would be greatly contributed for the detection of germline chimerism. Mixed- sex chimeras which contained both male and female cells were produced to investigate the developmental fate of male and female cells in ovary and testes. The sex combinations of donor and recipient of the resulting chimeras were following 4 pairs; (1) chimeras (ZZ/ZZ) produced by a male donor (ZZ) and a male recipient (ZZ), (2) chimeras (ZW/ZW) produced by a female donor (ZW) and a female recipient (ZW), (3) chimeras (ZZ/ZW) Produce by a male donor (ZZ) and a female recipient (ZW), (4) chimeras (ZW/ZZ) produced by a female donor (ZW) and a male recipient (ZZ). It was found that genetically male avian germ cells could differentiate into functional ova and that genetically female germ cells can differentiate into functional spermatozoa in the gonad of the mixed- sex chimeras. An ability for introduction of exogenous DNA into the PGCs from stage X blastoderms were analyzed. Two reporter genes, SV-$\beta$gal and RSV-GFP, were introduced into the PGCs. Expression of bacterial/gal was improved by complexing DNA with liposome detectedcc in 75% of embryos at 3 days embryos. At the embryos incubated for 1 day, expression of the GFP was observed all the embryos. At day 3 of incubation, GFP was detected in about 70% of the manipulated embryos. In case of GFP, expression of the transgene was detected in 30 %e of the manipulated embryos. These results suggested that the cells is one of the most promising vectors for transgenesis. The established strategy should be very powerfull for application to poultry biotechnology.

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Effects of Lipopolysaccride-induced Stressor on the Expression of Stress-related Genes in Two Breeds of Chickens (Lipopolysaccride 감염처리가 닭의 품종간 스트레스연관 유전자 발현에 미치는 영향)

  • Jang, In Surk;Sohn, Sea Hwan;Moon, Yang Soo
    • Korean Journal of Poultry Science
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    • v.44 no.1
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    • pp.1-9
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    • 2017
  • The objective of the present study was to determine the expression of genes associated with lipopolysaccharide (LPS)-induced stressor in two breeds of chickens: the Korean native chicken (KNC) and the White Leghorn chicken (WLH). Forty chickens per breed, aged 40 weeks, were randomly allotted to the control (CON, administered the saline vehicle) and LPS-injected stress groups. Samples were collected at 0 and 48 h post-LPS injection, and total RNA was extracted from the chicken livers for RNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. In response to LPS, 1,044 and 1,193 genes were upregulated, and 1,000 and 1,072 genes were downregulated in the KNC and WLH, respectively, using a ${\geq}2$-fold cutoff change. A functional network analysis revealed that stress-related genes were downregulated in both KNC and WLH after LPS infection. The results obtained from the qRT-PCR analysis of mRNA expression of heat shock 90 (HSP90), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), activating transcription factor 4 (ATF4), sterol regulatory element-binding protein 1 (SREBP1), and X-box binding protein 1 (XBP1) were confirmed by the results of the microarray analysis. There was a significant difference in the expression of stress-associated genes between the control and LPS-injected KNC and WLH groups. The qRT-PCR analysis revealed that the stress-related $HSP90{\alpha}$ and HMGCR genes were downregulated in both LPS-injected KNC and WLH groups. However, the HSP70 and $HSP90{\beta}$ genes were upregulated only in the LPS-injected KNC group. The results suggest that the mRNA expression of stress-related genes is differentially affected by LPS stimulation, and some of the responses varied with the chicken breed. A better understanding of the LPS-induced infective stressors in chicken using the qRT-PCR and RNA microarray analyses may contribute to improving animal welfare and husbandry practices.

The Evaluation of External Radiation Exposure dose rate for Radium-223 Dichloride (Radium-223 Dichloride의 외부 방사선량의 평가)

  • Cho, Seong Wook;Yoon, Seok Hwan;Seung, Jong Min;Kim, Tae Yub;Im, Jeong Jin;Kim, Jin Eui
    • The Korean Journal of Nuclear Medicine Technology
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    • v.20 no.1
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    • pp.28-31
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    • 2016
  • Purpose $^{223}Ra-Dichloride$ is used for the medicine of castration-resistant prostate cancer (CRPC) and which emits ${\alpha}-ray$ of 28 Mev that is used for therapy. However $^{223}Ra-Dichloride$ emits ${\beta}-ray$ of 3.6% and ${\gamma}-ray$ of 1.1%(80,156,270 keV) aside from ${\alpha}-ray$ in decay. Therefore we would like to evaluate external radiation expose dose rate of ${\gamma}-ray$ of $^{223}Ra-Dichloride$. Materials and Methods We calculated external radiation expose dose rate using ${\gamma}-constant$ of $^{223}Ra-Dichloride$, $^{99m}Tc$ based on Health physics(2012). $^{223}Ra-Dichloride$ of 3.5 MBq and $^{99m}Tc-MDP$ of 740 MBq were applied. external radiation expose dose rate 15 times from 1m by survey meter. Results ${\gamma}-contant$ of $^{223}Ra$, $^{99m}Tc-MDP$ from 1m distance based on Health physics(2012) is 0.0469, 0.0215. calculated value of external radiation expose dose rate was $16{\mu}Sy$, $34{\mu}Sy$ which activity is $^{223}Ra-Dichloride$ of 3.5 MBq and $^{99m}Tc-MDP$ of 740 MBq from 1 m and measured mean value of 1 m was $0.7{\mu}Sy/h$, $18{\mu}Sy/h$. Conclusion ${\gamma}-constant$ of $^{223}Ra$ is higher than $^{99m}Tc$ based on Health physics(2012). however calculated maximum external radiation expose dose rate of $^{223}Ra-Dichloride$ is lower than $^{99m}Tc$ due to actually used quantity of activity of $^{223}Ra-Dichloride$ is small. measured value of $^{223}Ra-Dichloride$ is also lower than $^{99m}Tc-MDP$. Therefore external radiation expose dose rate of ${\gamma}-ray$ of $^{223}Ra-Dichloride$ is very low.

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Hepatitis B Virus-Induced TNF-a Expression in Hepa-lc1c7 Mouse Hepatoma Cell Line (마우스 Hepa-1c1c7 세포주에서 B형 간염 바이러스에 의한 tumor necrosis factor-a의 발현 유도)

  • Yea Sung Su;Jang Won Hee;Yang Young-Il;Lee Youn Jae;Kim Mi Seong;Seog Dae-Hyun;Park Yeong-Hong;Paik Kye-Hyung
    • Journal of Life Science
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    • v.15 no.1 s.68
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    • pp.38-44
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    • 2005
  • Infection with hepatitis B virus (HBV) is a major health problem worldwide. Although a tremendous amount has been known about HBV, there have been obstacles in the study of HBV due to the narrow host range of HBV limited to humans and primates. In the present study, we investigated the susceptibility to HBV infection of mouse hepatoma cell line, Hepa-1c1c7. In addition, based on that human hepatocytes infected by HBV increase the expression of the pro-inflammatory cytokine TNF-a, the inducibility of TNF-a expression by HBV in the cells was determined. HBV surface antigen (HBsAg) secretion was measured by the microparticle enzyme immunoassay and steady state mRNA expression was analyzed by quantitative competitive RT-PCR. Transient transfection of Hepa-1c1c7 cells with HBV expression vector resulted in a dose-dependent induction of TNF-a expression. Infection of Hepa-1c1c7 cells with the serum of HBV carrier also increased TNF-a mRNA expression. Both in the transfected and infected cells, HBV mRNA was expressed and significant HBsAg secretion was detected. There was no significant variation in $\beta-actin$ mRNA expression by HBV. These results demonstrate that HBV is infectious to Hepa-lc1c7 in vitro and the viral infection induces TNF-a expression, which suggests that Hepa-lc1c7, a mouse hepatoma cell line, may be a possible model system for analysis of various molecular aspects of HBV infection.

Hypoglycemic Effect of Chlorella sp. CMS-1 Hot Water Extract on Streptozotocin-Induced Diabetic Rats (Streptozotocin-유발 당뇨쥐에 대한 클로렐라 열수 추출물의 혈당 강하 효과)

  • Kim, Jung-Wook;Cha, Jae-Young;Heo, Jin-Sun;Jin, Hyun-Jin;Cho, Young-Su
    • Journal of Life Science
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    • v.18 no.11
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    • pp.1584-1591
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    • 2008
  • The effect of Chlorella hot water extract (CE) on hyperglycemia in streptozotocin- induced diabetic rats has not been studied. Therefore, hypoglycemic effect of CE in type I streptozotocin- induced diabetic rats was studied. Rats were fed a semisynthetic diet supplemented with either 3% (the STZ+CE3) and 6% (the STZ+CE6) CE or no supplement the Normal and the STZ-Control rats for 4 weeks. The concentrations of fasting and non-fasting blood glucose were higher in the STZ-Control rats than in the Normal rats, but this rise was lowered in the STZ+CE3 and the STZ+CE6 rats. Serum insulin concentrations were decreased with STZ injection, however, the decreased levels were almost restored to the Normal level with CE supplementation. The increased serum fructosamine levels associated with hyperglycemia were decreased with the CE treatment. The morphology of pancreatic islets in the Normal rat was round and maintained a typical arrangement. The STZ-Control pancreatic beta-cells were found to have significant swelling and severely morphological damaged, however, pancreatic tissue damage by STZ in the CE-supplemented diet group was ameliorated. This study shows that Chlorella hot water extract had a hypoglycemic effect on the STZ-diabetic rats via either increased insulin secretion during recovery or the prevention of STZ-induced pancreatic damage.

Growth Effect of Oncorhychus masou by Recombinant Myostatin Prodomain Proteins Derived from Fish (어류 유래 마이오스타틴 프로도메인 단백질에 의한 시마연어(Oncorhychus masou) 성장효과)

  • Kim, Jeong-Hwan;Lee, Sang-Beum;Cho, Mi-Jin;Ahn, Ji-Young;Lee, Suk-Keun;Hong, Sung-Youl;Seong, Ki-Baik;Jin, Hyung-Joo
    • Journal of Life Science
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    • v.21 no.8
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    • pp.1149-1155
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    • 2011
  • Myostatin (MSTN) belongs to the transforming growth factor-${\beta}$ superfamily or growth and differentiation factor 8 (GDF-8), and functions as a negative regulator of skeletal muscle development and growth. Previous studies in mammals have suggested that myostatin knock-out increased muscle mass and decreased fat content compared to those of the wide type. Recently, several studies on myostatin have beenconducted on the block myostatin signal pathway with myostatin antagonists and the MSTN regulation with RNAi to control myostatin function. This study was performed to analyze growth and muscle alteration of Oncorhychus masou by treatment with recombinant myostatin prodomains derived from fish. We designed myostatin prodomains derived from P. olivaceus (pMALc2x-poMSTNpro) and S. schlegeli (pMALc2x-sMSTNpro) in a pMALc2x expression vector, and then purified the recombinant proteins using affinity chromatography. The purified recombinant proteins were treated in O. masou through an immersion method. Recombinant protein treated groups did not show a significant difference in weight, protein, or lipid composition compared to the control. However, there was a difference in the average number and area for histological analyses in the muscle fiber. At twelve and twenty-two weeks from the initial treatment, there were differences in averagefiber number and area between the 0.05 mg/l treated-group and the control, but the numbers were similar to those of the control during the same time period. At twelve weeks, however, 0.2 mg/l treated-group had an increase in average fiber number and decrease in average fiber area compared to the control. At twenty-two weeks, the pMALc2x-sMSTNpro 0.2 mg/l treated-group was induced and showed a decrease in average fiber number and increase in average fiber area. The results between twelve and twenty-two weeks showed that the fiber numbers had decreased, whereas average fiberarea had increased due to sMSTNpro. It is understood that the sMSTNpro induced only hyperplasia at twelve weeks, after which it induced hypertrophy. Recombinant myostatin prodomains derived from fish may induce hyperplasia and hypertrophy in O. masou depending upon the time that has elapsed.

Analysis of Hemolytic Microflora from the Ark Shell (Scapharca broughtonii) (패류(Scapharca broughtonii) 유래의 용혈활성 미생물 다양성 분석)

  • Kim, Dong-Gyun;Nam, Bo-Hye;Kong, Hee-Jeong;Kim, Woo-Jin;Kim, Bong-Seok;Jee, Young-Ju;Lee, Sang-Jun;Jung, Choon-Goo;Kong, Mi-Sun;Kim, Young-Ok
    • Journal of Life Science
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    • v.22 no.5
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    • pp.642-649
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    • 2012
  • The southern coast of Korea is important for the ark shell ($Scapharca$ $broughtonii$) aquaculture, but the productivity was rapidly reduced during the previous decade by mass mortality. To overcome this economic loss, investigations only focused on environmental factors, and microbiological researches were performed insufficiently. In this study, two sites (Gangjin and Jinhae bay) were selected for their high and low rate of mortality, respectively, and the existence of microflora from underwater sediments in the bodies of $S.$ $broughtonii$ was analyzed. We screened the whole body of each sample and chose unique colonies, which exhibit alpha- and beta-hemolytic activity, for identification. The microflora in $S.$ $broughtonii$ was less variable than sediments, and restricted species were isolated. We identified 17 genera of 88 species and 16 genera of 64 species from the two bays, respectively. A major proportion was comprised of $Bacillus$ species, with the $Bacillus$ $cereus$ group being the most common species among the $Bacillus$ strains, while $Paenibacillus$, $Lynsilbacillus$, and $Vibrio$ species were the second most abundant species. At the genus level, there were no significant microbial differences between the two coastal regions. 64 species were isolated from rare site (Jinhae bay), but more species (88) with greater variety were isolated from the frequent site (Gangjin bay). Therefore, it was assumed that the cause of mass mortality lay in the difference in specie-level diversity, and conducting investigations on the diagnosis of pathogenic species by challenging tests using isolated unique species.

Quality and Antioxidant Properties of Iced Cookie with Black Tomato (Lycopersicum esculentum) Powder (흑토마토 분말을 첨가한 냉동 쿠키의 품질 및 항산화 특성)

  • O, Hyeonbin;Choi, Byung Bum;Song, Ka-Young;Zhang, Yangyang;Kim, Young-Soon
    • The Korean Journal of Food And Nutrition
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    • v.29 no.1
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    • pp.65-72
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    • 2016
  • Black tomato (Lycopersicum esculentum) is known to have more ${\beta}-carotene$, lycopene, and vitamin C than general red-colored tomatoes. In this study, we evaluated the quality properties, antioxidant activities and sensory characteristics of black tomato cookies. Cookies were prepared by replacing 0, 1, 3, 5, and 7% of flour with black tomato powder. Density of black tomato cookies tended to be decreased between control (1.20) and 3% added groups (1.12). pH value was decreased from control (6.66) to 7% added group (5.16). Spread factor and loss rate were increased with increasing amounts of black tomato powder. Hardness was gradually increased from $107.77g/cm^2$ in control to $170.50g/cm^2$ in 7% added group. Color measurement indicated that L-value (brightness) was highest in control (70.46) and lowest in 7% added group (45.23); whereas, a-value (redness) increased while b-value (yellowness) tended to decrease with increasing amounts of black tomato powder. Total polyphenol contents and DPPH radical scavenging activities were directly proportional to the amount of black tomato powder. Consumer preference scores in color and flavor of black tomato powder added group were higher than those of control. Characteristic strength test was not significantly different among the groups. Overall, the results indicated that adding 5% black tomato powder is desirable for making black tomato cookies.

Effects of Soy Isoflavone on the Bone Metabolism in Ovariectomized Growing Rats (콩 이소플라본이 난소를 절제한 성장기 흰쥐의 골격대사에 미치는 영향)

  • Kim, Young-Kyoung;Lee, Heon-Ok;Om, Ae-Son
    • Korean journal of food and cookery science
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    • v.21 no.5
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    • pp.725-732
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    • 2005
  • This study attempted to investigate if the soy isoflavone, genistein, influences bone metabolism in ovariectomized, 4-week-old female Wister rats. All the rats were divided into sham (SH) and ovariectomized (OVX) groups consisting of OVX-17${\beta}$-estradiol($10\;{\mu}g/kg$ b.w.), OVX-1mg or 5 mg or 10mg of genistein/kg b.w. The rates were allowed ad libitum access to foods and water for 8 weeks. The Results showed that body weight had significantly increased in the OVX group compared to the SH group (p<0.05) and was not different among the OVX-GEN and OVX-ES groups and the OVX group. The liver and uterus weights in the OVX groups were slighter than those in SH group (p<0.05). The kidney weight in the OVX groups was decreased compared to in that in the SH group but was not significantly different among all OVX groups. Femoral length in the OVX groups was longer than in the SH group and was not different. Rats in the OVX groups had higher creatinine levels than those in the SH group and hydroxyproline level did not differ significantly among any of the groups. Serum ALP activity and Ca in bone, feces, urine and serum did not change among the groups. Bone mineral density (BMD) decreased in the OVX groups compared to the SH group and was slightly increased by feeding genistein (p>0.05). Breaking force and stiffness did not change by ovariectomy and feeding genistein. Hence, these results suggested that estrogen may affect bone mineralization in growing rats and that genistein be may involved in the prevention of bone loss. However, more studies are needed to identify the proper mechanism of genistein and bone formation.

Anti-oxidative and Cytoprotective Effect of Ursodeoxycholic Acid, an Active Compound from the Bear's Gall, in Mouse Microglia (생쥐 뇌소교세포주에서 웅담추출활성성분(우르소데옥시콜린산)의 항산화 및 세포보호효과)

  • Joo, Seong-Soo;Kim, Seong-Kun;Yoo, Yeong-Min;Ryu, In-Wang;Kim, Kyung-Hoon;Lee, Do-Ik
    • Korean Journal of Food Science and Technology
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    • v.38 no.3
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    • pp.452-455
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    • 2006
  • The in vitro cytoprotective and anti-oxidative effects of ursodeoxycholic acid, a major active compound from bear's gall were investigated in mouse brain microglia. In the present study, we wished to scrutinize the potential role of UDCA as an anti-neurodegenerative agent in neurodegenerative disease such as Alzheimer's disease. This concept was supported by the multiple preliminary studies in which UDCA has an anti-inflammatory effect in microglial cells. In the study, we found that $7.5\;{\mu}g/mL$ UDCA was effective in the protection of cells from $H_2O_2$ damage, a reactive oxygen, and the resuIt was coincided with the anti-apoptotic effect in DAPI staining. Moreover, the metal-catalyzed oxidation study showed that UDCA has antioxidant effect as much as ascorbic acid at $50{\sim}100\;{\mu}g/mL$. In conclusion, these study results suggested that neuro-degenerative diseases such as Alzheimer's disease probably caused by over-expressed beta amyloid peptide in elderly people can be controled by UDCA through an anti-inflammatory, anti-oxidative and anti-apoptotic effect. The evidences showed in the study may be references for more in-depth in vivo and clinical studies for a candidate of anti-neurodegenerative therapy in the near future.