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Rat H-Y 항체에 의한 생쥐 Embryo 의 성 조절에 관한 연구 1 . H-Y 항체의 처리가 생쥐 상실배 (桑實胚) 의 발달에 미치는 영향 및 세포독성시험
정장용(J . Y . Jeung),박충생(C . S . Park),박희성(H . S . Park) 한국축산학회 1989 한국축산학회지 Vol.31 No.8
This experiment was carried out to develop a new technique by immunological method. H-Y antiserum was prepared in inbred Wistar female rats by repeated immunization with new-born testis supernatant and spleen cells from males of the same strain. The activity of H-Y antibody in antiserum was tested by cytotoxicity and biological tests. The results obtained in this experiment. are as follows: In the sperm cytotoxicity test it was showed about 70% of the sperm were dead in the 1/2 to 1/8 dilution of H-Y antiserum immunized with spleen cells, new-born testis or the compound of both antigens. As the dilution increased, the death rate of sperm decreased markedly. H-Y antibody absorbed with female-rat spleen cells showed higher death-rate of sperm than that with male-rat spleen cells. The normal female-rat serum showed the sperm death-rate of 14.6 to 27.9% irrespective of the dilution rate. The difference between the two sperm death-rates was significant. The embryos cultured in the medium of complement and H-Y antiserum immunized with male-rat spleen, new-born testis or the compound of both antigens showed the lysis-rates of 48.9, 50.0 and 46.3% respectively. There was no significant difference among each lysis-rate. But the lysis-rate of the embryos cultured in the medium of complement and normal female rat serum was 5.1% and it was markedly different from the above lysis rates(p $lt;0.001).
Rat H - Y 항체에 의한 생쥐 Embryo 의 성 조절에 관한 연구 Ⅲ. H - Y 항혈청에 의한 BALB c 생쥐 상실배의 성 판별
정장용(J . Y . Jeung),박충생(C . S . Park),박희성(H . S . Park) 한국축산학회 1989 한국축산학회지 Vol.31 No.10
This experiment was carried out to develop a new technique by identifying XX-bearing embryos prior to implantation by immunological method. H-Y antiserum was prepared in inbred Wistar female rats by repeated immunization with spleen cells from males of the same strain. The reactivity of H-Y antibody was confirmed by culturing mouse embryos in the medium containing H-Y antiserum and complement obtained from the guinea pig. The optimal condition for the activity of H-Y antibody was also investigated by culturing embryos under the various conditions of equilibration times, complement concentrations and various media. The results obtained in this experiment are as follows: When the embryos were cultured in the medium of H-Y antiserum and complement which was given the equilibration time of less than 30 minutes in CO₂ incubator, the lysis-rate of embryo was 89.3%. The embryo lysis-rates in the equilibration time of 1-1.5, 3-3.5, or 24-26 hours were 48.1, 47.7 and 48.2%, respectively. When the concentration rate of complement to H-Y antiserum varied from 0.25 - 4.0, the lysis-rate of embryo was 43.2 to 52.7%. The concentration rate of complement did not influence the lysis-rate of embryos. The meda of D-PBS + 0.3% BSA, D-PBS + 20% FCS, Ham`s F-10 + 0.3% BSA and Ham`s F-10 + 20% FCS showed the embryo lysis-rate of 46.4, 57.4, 49.3 and 49.1%, respectively. The culture media used in this experiment did not show any significant difference in the embryo lysis-rate. After the embryos were cultured to the late blastocyst in the media of D-PBS + NGPS + H-Y antiserum or D-PBS + NGPS + normal female rat serum the normally developed embryos were selected and transferred to the pseudo pregnant recipients. The percentages of their female offspring were 82.3%(14/17) in H-Y antiserum treatment and 53.6%(15/28) in normal serum treatment and showed a significant difference between the two treatments(p $lt;0.001).
박희성(H . S . Park),정장용(J . Y . Chung),박충생(C . S . Park) 한국축산학회 1992 한국축산학회지 Vol.34 No.1
The present study was carried out to determine the effect of nuclear transplantation on the postnatal development and the reproductive performance in mice. The conception rate of recipient mice following the transfer of blastocysts reconstituted with nuclei donor of 2-to 4-cell stage was similar(P$gt;0.05) to the result with intact blastocysts(61.6%), but the conception rate with blastocysts from nuclei donor of 8-cell stage was significantly(P$lt;0.05) decreased to 20.0%. On the basis of` their body weights at weaning and puberty, conception rate and litter size at birth, the postnatal development and reproductive performance, male and female mice produced by transfer of nuclear transplanted blastocysts were comparable to those of the control mice which were produced by transfer of is vitro developed blastocysts.
박희성(H . S . Park),박충생(C . S . Park) 한국축산학회 1991 한국축산학회지 Vol.33 No.12
The present study was carried out to improve the technology of nuclear transplantation using mouse embryos as a model and to produce nuclear transplanted mice by transferring reconstituted mouse embryos to recipient females. The donor embryos of two- four- and eight-cell stage were preserved for 4 to 10 hours at 4℃ in order to synchronize the cell cycles with those of the recipient embryos from mice raised in the same light-controled room, and the resistance of donor embryos against the low temperature was also examined. Single nucleus from two-. four- and eight-cell embryos was transplanted into the enucleated two-cell embryos by micromanipulation and Sendai virus-mediated fusion. The fusion of nuclei with recipient cytoplasm and the development of reconstituted embryos in vitro and in vivo were examined. The rates of enucleation, nucleus injection into the perivitelline space of recipient embryos and nucleus fusion with recipient cytoplasm were similar(P$gt;0.05) between the cell stages of nuclear donor embryos. The development in vitro to blastocysts of the nuclear transplanted embryos with nuclei of 2-. 4- and 8-cell donor embryos preserved at 4℃ was 74.0, 58.0 and 44.8%. respectively, and was significantly(P$lt;0.05) different between the cell stages of nuclei donor embryos. The duration for blastocoele formation following in vitro culture of embryos was shorter(P $lt;0.05) in intact embryos of 2-cell stage, compared with the reconstituted embryos with nuclei from donor embryos of 2-, 4- and 8-cell stage. The conception rate of recipient mice following embryo transfer was similar between intact and nuclear transplanted embryos with donor nuclei of 2- to 4-cell stage. but was decreased significantly (P$lt;0.05) following transfer of embryos with donor nuclei of 8-cell stage. The percentage of embryos which developed to term following embryo transfer was significantly(P$lt;0.05) decreased in nuclear transplanted blastocysts. compared with the in vitro developed blastocysts. About 50% of transferred intact embryos were developed in vivo to term, but the percentage of embryos which developed to term was decreased significantly (P$lt;0.05) in all the nuclear transplanted embryos and to 13.5% in the reconstituted embryos with donor nuclei of 8-cell stage. From the above results obtained it was concluded that preservation of donor embryos at low temperature for a relatively short period had no adverse effect on the subsequent development in vitro or in vivo of the nuclear transplanted embryos.
생쥐 8- 세포기 수정란의 핵이식에 의한 복제산자의 생산
최상용(S . Y . Choe),박희성(H . S . Park),이효종(H . J . Lee),박충생(C . S . Park) 한국축산학회 1992 한국축산학회지 Vol.34 No.2
The present study was carried out to develop a technology of cloning mammalian embryos using mouse embryos as a model and to produce cloned offsprings by transfer of reconstituted embryos to recipient females. Single nucleus from eight-cell embryos was transplanted into the enucleated two-cell embryos by micromanipulation and Sendai virus-mediated fusion. The fusion of nuclei with recipient cytoplasm and the development of reconsitituted embryos in vitro as well as in vivo were examined. The results obtained were summarized as follows: A total of 95(59.3%) nuclei from 20 donor embryos of 8-cell stage was injected successfully into the enucleated 2-cell recipient embryos, of which octa-. hepta-, hexa-, penta- and quadruplets were 0. 2, 3, 3 and 12 sets, respectively. The success in nuclei injection was lower(P$lt;0.05) from 8-cell donor embryos than 4-cell or 2-cell donor embryos. Among the embryos injected with nuclei a total of 81(85.2%) embryos were fused successfully with donor embryos of 8-cell stage. From donor embryos of 8-cell stage, 2 hexa-, 2 penta-, 11 quadru- and 5 triplets were fused successfully. A total of 46(39.l%) reconstituted embryos from 8-cell donor nuclei were developed in vitro to blastocysts, of which penta-, quadru- and triplets. twins and singles were 1, 2, 6, 6 and 3 sets, respectively. The development in vitro to blastocysts of the reconstituted embryos was lower (P$lt;0.05) from 8-cell donor embryos than 4- or 2-cell donor embryos. A total of 13(27.l%) cloned live youngs from transfer of cloned embryos using 8-cell donor embryos were produced, of which triplets, twins and singles were 2, 3 and 1 sets, respectively. The percentage of cloned youngs produced from transfer of cloned embryos using 8-cell donor embryos was lower(P$lt;0.05) than 4- or 2-cell donor embryos. The mean number of cloned live youngs produced per 8-cell donor embryo used for nuclei transplantation was 0.81.