• 한국수정란이식학회지
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• 제28권3호
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• pp.185-191
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• 2013

The purpose of this study is to improve production efficiency of vitrified-thawed transgenic bovine embryos. Transgenic bovine embryos were produced by injection of FIV-GFP lentiviral vector into perivitelline space of in vitro matured MII stage oocytes, and then in vitro fertilization. EGFP-expressing transgenic bovine blastocysts were cultured in serum-containing and serum-free medium. These blsatocysts were vitrified by pull and cut (PNC) container made with 0.25 cm plastic straw. Results indicate that total developmental rates of normal IVF embryo cultured in serum-containing and-free medium into blastocyst were not significantly different (22.3 vs 21.5%) and those of GFP-expressing transgenic bovine embryo into blastocyst showed no significant difference between serum-containing (13.9%) and-free medium (13.1%). However, developmental rate of GFP transgenic embryo was significantly (P<0.05) lower than its of normal IVF embryos. In additional study, we vitrified GFP transgenic normal bovine blastocysts using PNC vitrification method. Survival rate of vitrified-thawed GFP transgenic blastocyst (23.1%) was significantly (P<0.05) lower than its of normal blastocysts (68.9%). Although, survival rate of vitrified-thawed GFP transgenic blastocyst was lower than its of normal blastocyst, our result may suggested that PNC vitrification method is feasible to cryopreserve transgenic embryos. Our next plan will be the production of GFP express transgenic bovine derived from vitrified-thawed embryos using PNC method.

• Fisheries and Aquatic Sciences
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• 제18권1호
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• pp.81-88
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• 2015

We investigated factors affecting primary cultures of Pacific abalone Haliotis discus hannai ovary-dissociated cells to identify general aspects of their early-phase culture. Ninety-seven cell populations derived from 30 individuals were cultured in different media with varying compositions of medium supplements, and initial attachment, subculture, and survival for ${\geq}10$ weeks were assessed according to medium composition and individual. We also examined the time required for subculture and the rate of cell death according to both culturing period and passage number within 10 weeks. A lack of fetal bovine serum (FBS) and hemolymph significantly inhibited the growth of cultured cells, while we detected no significant effect of medium composition on initial cell attachment. Through data reallocation, with the omission of data from cell populations cultured in FBS-free and hemolymph-free media, we showed that growth inhibition was also affected by individual differences among the abalones used. During the culture, we observed four different types of cell morphology. Moreover, considerable time was required for subculture-18.4 and 19.5 days for first and second subcultures, respectively-and cell death did not occur within 30 days or for passage 0. Our results will provide valuable information for developing universal cell culturing guidelines in abalone species and suggest the feasibility of culturing abalone ovary-dissociated cells.

• Asian-Australasian Journal of Animal Sciences
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• 제18권11호
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• pp.1557-1563
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• 2005

Ovarian follicular fluid contains both stimulatory and inhibitory agents that influence the growth and maturation of oocyte. In the present study, an attempt was made to isolate and study the biological properties of ovarian follicular fluid peptide(s) in buffaloes. Bubaline ovarian follicular was made steroid- and cell-free. A protein fraction was obtained by saturation (30-35% level) of the follicular fluid with ammonium sulfate. The protein fraction was purified with Sephadex-G 50 gel filtration chromatography and a single peak was obtained in the eluant volume, which was lyophilized. SDS-PAGE of the lyophilized fraction revealed a single band and the molecular weight of the peptide was 26.6 kDa. The peptide stimulated the cumulus cell expansion and in vitro maturation rate of oocytes in buffaloes in a dose dependent manner when it was incorporated at different dose levels (0, 10, 25, 50, 100 and 1,000 ng $ml^{-1}$ of maturation medium). The basic culture medium consisted of TCM 199 with Bovine serum albumin (0.3%). The in vitro maturation rates were comparable to those obtained with a positive control medium (TCM 199+20 ng EGF $ml^{-1}$+steer serum (20%)). Further purification and biological assays may throw more light on the nature and functions of this peptide.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.335-339
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• 2003

본 연구에서는 연골조직으로부터 세포를 분리한 후 10%의 혈청 첨가 배지와 무혈청 배지에서의 세포 성장속도, GAGs 합성 및 Col. II의 발현을 확인하였다. EGF를 첨가한 무혈청 배지를 연골세포의 증식에 매우 효과적이었으나 GAGs 합성 및 Col. II 의 합성을 저해하였다. 또한 무혈청 배지에서 배양한 세포를 차후 신체내로 이식한다면 연골세포의 특성인 Col. II를 재합성할 수 있음을 간접적으로 확인하였다. 이는 무혈청 배지를 이용하여 평판배양 시 짧은 기간 내 연골세포 치료제로서 가능한 세포수를 얻기 위한 모델로서 유용할 것으로 생각된다. 또한 계대배양에 따른 분화 및 형태의 변화 등의 문제점들은 생분해성 지지체와 연계하여 해결할 수 있을 것으로 생각되며, 차후 위의 SFM을 이용한 3D 배양에 대한 연구를 수행할 예정이다.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Asian-Australasian Journal of Animal Sciences
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• 제9권2호
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• pp.123-126
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• 1996

Sixteen Harnai males were used to evaluate the influence of varying levels of fourwing saltbush hay on feed and water intakes as well as the blood serum mineral status in a completely randomized design. The animals were grouped randomly into four, with four animals in each. The four groups were randomly allotted low, medium, high and very high levels of saltbush hay supplementation in addition to wheat straw. The animals were given fresh water at free of choice. Weekly body weight was recorded for each individual animal. Blood serum was collected for mineral contents. The experiment lasted for eight weeks. The inclusion of saltbush leaves in the diet showed a non-significant effect on the total dry matter intake. There has been a significant increase in the water intake when very high levels of saltbush were included in the ration. Lower levels showed no effect on the water intake. The animals maintained their body weight from week 1 to week 8. No treatment by weeks interactions on the potassium and sodium levels were detected. However higher levels of saltbush increased significantly the potassium and sodium contents in the serum. Calcium contents were significantly(p<0.01) lowered with the inclusion of saltbush leaves in the diet. Whereas Phosphorous contents showed an increasing(p<0.05) trend with the higher levels of saltbush. No clinical or sub-clinical toxicological symptoms were observed in the sheep with the higher mineral contents.

• Preventive Nutrition and Food Science
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• 제1권1호
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• pp.80-86
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• 1996

Parathyroid hormone(PTH) is known to stimulate bone resorption and to inhibit bone collagen synthesis. In contrast, as the evidence of stimulation of bone formation by PTH has recently been observed, the study on the role of PTH involved in osteoporosis draws remarkable attention. This study has dealt with the role of alkaline phoshatase(AP), a marker enzyme for bone formation and osteoblast action, Animals(BALS/cmice) were divided into three dietary groups(high and medium Ca and Ca-free) and hormones including PTH, calcitonin(CT), cholecalciferol(citamin D) were i.p. injected. AP in the serum and liver was measured using Sigma 221 alkaline buffer solutions containing 9mM of p-nitrophenyl phoshate. Enzume was reacted at 37$^{\circ}C$ for 10 minutes and the reaction was stopped by 1.8ml of 0.1N NaOH and measured at 410nm. We found that serum and liver AP activity was increased by low dietary Ac. Compared to the control, and serum Ap activity was enhanced by PTH and vitamin D regardless of the dietary Ca. On the other hand, liver AP activity was inhibited by OTH and vitamin D at all levels of dietary Ca. CT inhibited the action of PTH and vitamin D in the serum. But, the inhibition of PTH and vitamin D action by CT was not observed in the liver, unlike in the case of serum.

• KSBB Journal
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• 제21권4호
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• pp.248-254
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• 2006

본 연구에서는 CHO 세포를 이용해 세포를 별도의 적응기간 없이 무혈청 배지에서 배양했을 때 세포의 증식이 중단되는 원인을 찾고 배지 첨가 성분을 통해 이를 개선하고자 했다. 현재 개발된 무혈청 배지는 아직까지 혈청을 대체할 만한 성분을 포함하고 있지 않다. 때문에 무혈청 배지에 적응되지 않은 비적응 세포의 경우 계대 배양에 한계가 있다. 이런 한계가 나타나는 원인은 다양할 것으로 생각이 되지만 혈청의 부재로 인해 세포가 받게 되는 스트레스와 그로 인한 세포주기의 정지가 가장 근본적인 원인으로 생각된다. 무혈청 배지에서 세포가 받는 스트레스의 정도를 알아보고 배양 환경과 첨가물에 따른 ROS 농도의 변화를 측정하기 위해 배지와 세포의 ROS 농도를 측정하였다. ROS 농도를 측정한 결과 무혈청 상태에서 세포내 ROS가 엄청난 양으로 증가하는 것을 알 수 있었다. 이것은 혈청이 항산화능력을 갖고 있어서가 아니라 세포가 무혈청 환경에서 극심한 스트레스상태에 놓이기 때문인 것으로 생각된다. 이렇듯 증가한 ROS가 세포의 증식이 멈추게 되는 원인 중 하나로 생각되고, 항산화제를 첨가한 경우에도 증식력이나 ROS의 농도에 큰 차이가 없었던 것으로 미루어 보아 근본적으로 혈청과 같은 강력하게 증식을 촉진하는 성분을 배지에 첨가해야 할 것으로 여겨진다. ROS 이외에 세포의 증식이 멈추는 또 다른 원인으로 세포사멸의 여부를 확인했다. 무혈청 배지에서 배양한 적응세포와 비적응 세포 모두 특별한 세포사멸의 징후가 나타나지 않았다. 또한 무혈청 배지에서 증식이 멈춘 세포를 회수해 다시 혈청배지에서 배양한 경우 곧바로 증식력이 회복되기 때문에 대규모의 세포사멸은 발생하지 않는 것으로 생각된다. 위와 같은 현상들은 모두 혈청이 없기 때문에 발생하는 것으로 혈청을 대체할 수 있는 첨가물을 배지에 더해주면 세포의 증식이 개선될 것이다. 그래서 몇 가지 첨가물을 이용해 세포의 증식력에 변화가 나타나는지 알아보았다. 첨가물을 이용한 실험에서 IGF-I의 경우 장기간 배양에서 세포의 수를 안정적으로 유지하고 계대 횟수를 증가시키는 효과를 보였다. 이는 IGF-I이 어느정도 세포의 증식을 유지시켜주는 역할을 하기 때문인 것으로 생각된다. 무혈청 배지에서 비적응 CHO 세포의 계대 배양에 한계가 있는 것은 세포주기가 멈추기 때문인 것으로 생각된다. 세포주기가 멈추는 growth factor와 같이 세포의 증식을 지속적으로 유도할 수 있는 물질이 무혈청 배지에서는 부족하기 때문인 것으로 생각되고, IGF-I과 같은 첨가물을 통해 극복할 수 있는 문제라고 여겨진다.

• Journal of Microbiology
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• 제42권1호
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• pp.25-31
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• 2004

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8${\times}$l0$\^$6/ cells/$m\ell$ in a 500$m\ell$ spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-l strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15 % of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-l strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-l stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-l strain rose from 1.0${\times}$l0$\^$5/ to 1.5${\times}$l0$\^$6/ pfu/$m\ell$. The infected Sf9 cells could be subcultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-l strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-l strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.

• Journal of Ginseng Research
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• 제19권3호
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• pp.212-218
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• 1995

A human hepatoma cell line, hep G2, was used to investigate the mechanism of serum cholesterol reduction by ginseng total saponin, ginsenoside-$Rb_1$, - $Rb_2$, and non-saponin fraction (ether extraction). Hep G2 cells were incubated in 10 $\mu\textrm{g}$/ml of cholesterol containing serum free-RPMl1640 medium with various concentration of ginseng components. The amounts of cholesterol in Hep G2 cells were decreased to maximum 51% in total saponin or two ginsenoside-treated groups while there was 137% increase in cholesterol level of control group as compared with that of normal group. Nonsaponin groups did not show the same effect. In order to elucidate the observed changes in the amount of cholesterol, the activity of amyl CoA : cholesterol acyltransferase (ACAT) in groups showing remarkable reduction in cholesterol amount, i.e., total saponin 10-6%, ginsenoside-$Rb_1$ $10^{-4}$%, ginsenoside-$Rb_2$, $10^{-4}$%, and non-saponin fraction $10^{-4}$%, was assayed using [1-$^{-14}C$%]oleic acid as enzyme substrate. The activity of ACAT was increased in all groups tested as compared with that of control group except for non-saponin group cultured in water soluble cholesterol containing medium. The serum cholesterol lowering effects of ginseng components can partially be attributed to the increased hepatocellular ACAT activity.