• Title/Summary/Keyword: 생쥐 배아

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생쥐 초기배아에서 세포질내 요인에 의한 유전자 발현과 형태형성의 조절

  • 이동률
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 1998.07a
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    • pp.63-65
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    • 1998
  • 생쥐의 초기발생과정에서 배아 세포질의 제거로 nucleus/cytoplasm ratio를 증가시켰을 경우, 발생과 gene expression에는 영향을 미치지 않았으나 형태형성은 촉진되었다. 또, nucleus/cytoplasm ratio의 변화 없이 발생후기의 cytoplasm으로 일부 치환된 배아에서 발생과 gene expression, 형태형성은 모두 촉진되었다. 이상의 결과로 보아 초기배아의 gene expression과 형태형성과정에서 일어나는 time schedule의 조절은 nucleus/cytoplasm ratio가 직접적으로 영향을 미치지 않고 난자와 배아 내에 존재하는 cytoplasm factor에 의해 조절 받으며, 특히 형태 형성의 time schedule은 cytoplasmic factor와 이들에 의해 조절되는 물질의 상대적인 농도에 의해 조절될 것으로 여겨진다.

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Establishment and Characterization of Multipotent Germ Line Stem Cells (MGSCs) from Neonatal Mouse Testis (신생 생쥐 고환에서 기인한 다분화능 생식줄기세포주의 확립 및 특성 분석)

  • Han, Sang-Chul;Song, Haeng-Seok;Jun, Jin-Hyun
    • Clinical and Experimental Reproductive Medicine
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    • v.35 no.1
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    • pp.39-48
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    • 2008
  • Objective: The aim of this study was to investigate whether multipotent germline stem cells (MGSCs) can be established from neonatal mouse testis. Methods: Various cells containing MGSCs were collected from neonatal testis of ICR mice and allocated to plates for in vitro culture. After 7 days in culture, the cells were passed to a fresh culture plate and continuously cultured. From the third or fourth passage, the presumed MGSCs were cultured and maintained on mitomycin C-inactivated STO feeder cells. The MGSCs were cultured in a condition where mouse embryonic stem cells (ESCs) are cultured. Characteristics of the MGSCs were evaluated by RT-PCR, immunocytochemistry, alkaline phosphatase activity, karyotyping, and transmission electron microscopy. Results: Two MGSCs lines were established from 9 pooled sets of neonatal testicular cells. MGSCs colonies were morphologically undistinguishable from ESCs colonies and both MGSC lines as well as ESCs expressed undifferentiated stem cell markers, such as Thy-1, Oct-4, Nanog, Sox2 and alkaline phosphatase. Fine structure of undifferentiated MGSCs were similar to those of ESCs and 60% of MGSCs (12/20) had normal karyotype at passage 10. They were able to form embryoid bodies (EBs) and MGSC-derived EBs expressed marker genes of three germ layers. Conclusion: We could establish the MGSCs from neonatal mouse testis and they were differentiated to multipotent lineages of three germ layers. Molecular characteristics of MGSCs were similar to those of ESCs. Our results suggest a possibility that multipotent stem cells derived from testis, the MGSCs, could replace the ESCs in biotechnology and regenerative medicine.

Production of Chimeric Mice Following Transgenesis of Multipotent Spermatogonial Stem Cells (유전자변형 다분화능 정원줄기세포를 이용한 키메라 생쥐의 생산)

  • Lim, Jung-Eun;Eum, Jin-Hee;Kim, Hyung-Joon;Park, Jae-Kyun;Lee, Hyun-Jung;Lee, Dong-Ryul
    • Development and Reproduction
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    • v.13 no.4
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    • pp.305-312
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    • 2009
  • Multipotent spermatogonial stem cells (mSSCs), derived from uni-potent SSC, are a type of reprogrammed cells with similar characteristics to embryonic stem cells (ESCs). The aim of this study was to evaluate the potential for transgenesis of mSSC derived from outbred mice and the production of transgenic animal by the mSSC-insertion into embryo. mSSCs, established from outbred mice (ICR strain) in the previous study, were maintained and then transfected with a lenti-viral vector expressing green fluorescent protein (GFP), CS-CDF-CG-PRE. Embryonic stem cells (ESCs) were derived from inbred transgenic mice (C57BL/6-Tg (CAG-EGFP)) and were used as an experimental control. Transfected mSSCs were well proliferated in vitro and maintained their characteristics and normal karyotype. Ten to twelve mSSCs and ESCs were collected and inserted into perivitelline space of 8-cell mouse embryos, and then transferred them into uteri of poster mothers after an additional 2-days of culture. Percentage of mSSC-derived offsprings was 4.8% (47/980) and which was lower than those (11.7% (67/572)) of ESC-derived ones (P<0.05). However, even though different genetic background of mSSC and ESC origin, the production efficiency of coat-colored chimeric offspring in mSSC group was not different when compared it with ESC (6.4% (3/47) vs. 7.5% (5/67)). From these results, we confirmed that mSSC derived from outbred mice has a pluripotency and a potential to produce chimeric embryos or mice when reaggregatation with mSSC is performed.

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초기 배아의 분리할구에서 중기 염색체상 획득 방법에 대한 연구: 염색체 변이로 인한 착상전 유전자 진단에서 보인자와 정상 핵형 구분을 위한 연구

  • 임천규;전진현;민동미;변혜경;김진영;궁미경;강인수
    • Proceedings of the Korean Society of Embryo Transfer Conference
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    • 2002.11a
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    • pp.105-105
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    • 2002
  • 염색체의 구조적 이상으로 인한 습관성 유산과 기형아의 출산을 예방하기 위해 착상전 배아에서 할구를 분석하여 정상적인 핵형을 가진 배아만을 이식하는 착상전 유전자 진단 (preimplantation genetic diagnosis, PGD)의 성공적인 임상 적용이 보고되고 있으며, 그 적용 범위가 확대되고 있다. 그러나 일반적인 간기의 핵상을 이용한 PGD에서는 형광직접보합법 probe의 제약으로 보인자와 정상적인 핵형을 구분할 수 없는 단점이 있다. 따라서 본 연구에서는 보다 정확한 PGD를 위해 생쥐 배아를 이용하여 분리한 할구에서 중기 염색체상을 획득하기 위해 미세소관 (microtubule) 형성 저해제를 처리하였으며, 이를 통해 확립된 방법을 인간의 PGD에 적용하고자 하였다. 과배란이 유도된 ICR 생쥐에서 4- 또는 8-세포기 배아를 수획하여 colcemid, nocodazole, vinblastine을 각각 0.1, 0.5, 1.0, 5.0$\mu$M을 처리하고, hoechst 33342로 염색하여 핵상을 관찰하여 최적의 농도를 결정하였다. 또한 각 미세소관 형성 저해제를 혼합 처리하여 가장 높은 중기 염색체상을 획득할 수 있는 혼합 처리를 결정하였다. 이렇게 결정된 혼합 처리 방법을 인간의 체외 수정 및 배아 이식술에서 획득된 3PN 배아에 처리하여 중기 염색체를 획득하였다. Colcemid, nocodazole, vinblastine 모두 1 $\mu$M이 최적 농도임을 확인할 수 있었다 (각각 96.3%, 92.0%, 98,4%). 미세소관 형성저해제를 혼합 처리하였을 경우 nocodazole과 vinblastine (각각 1$\mu$M)을 혼합 처리했을 때 중기 염색체 획득률(97.3%)이 가장 높았다. 인간의 3PN 배아에 1$\mu$M의 nocodazole과 vinblastine을 혼합 처리한 후, 113개의 할구를 분석하여 44개(38.9%)의 할구에서 중기 염색체를 확인할 수 있었다. 본 실험 결과를 통해 중기 염색체를 획득하기 위하여 미세소관 형성 저해제를 처리하는 방법은 생쥐의 배아에서는 효과적이지만, 인간의 배아에서는 그 효율이 다소 낮음을 알 수 있었다. 그러나 이 방법을 개선하여 인간의 할구에서 중기 염색체의 획득률을 높이고, 이를 염색체의 구조적 이상에 대한 착상전 유전자 진단에 적용한다면, 보인자와 정상의 핵상을 구분하여 정상의 핵상만을 갖는 배아의 이식을 통하여 더욱 정확한 착상전 유전자 진단을 시행할 수 있으리라 사료된다.

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The Effects of Endocrine Disruptors on the Development of Mouse Preimplantation Embryos and the Regulation of Apoptotic Gene Repression (내분비계 장애물질이 착상전 생쥐 배아의 발생 및 Apoptosis 조절 유전자 발현에 미치는 영향)

  • 정경남;유정민;유성진;김주란;정철희;김현찬;강성구
    • Development and Reproduction
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    • v.6 no.1
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    • pp.25-30
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    • 2002
  • Endocrine disruptors have been reported to adversely affect reproduction and embryonic development in wild animals. One of the major abnormalities observed during early embryonic development is cellular fragmentation. In this study, we exposed mouse preimplantation embryos to PCB, BPA and DDT in vivo or in vitro. Embryos exposed to endocrine disrupter showed a variety of morphological abnormalities such as fragmentation, irregular blastomeres and cracked empty zonae pellucidae. To investigate the levels of gene expression related which genes contribute to apoptosis in preimplantation mouse embryos, we carried out the reverse transcription polymerase chain reaction to assess mRNA levels far apoptotic gene. Bcl-2, bad and bax expression levels were compared between control group and endocrine disrupter treated group. Expression level of bcl-2 gene tended to be lower in the treated group than control while expression levels of bad and bax genes were higher in the treated group. Results of this study may provide a useful tool for rapidly screening developmental toxicants in preimplantation embryos exposed to endocrine disruptors in vivo or in vitro.

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Comparison of Vitrification and Slow Freezing for the Cryopreservation of Mouse Pronuclear Stage Embryos (생쥐 전핵기 배아 냉동보존에서 완만동결과 유리화동결의 비교)

  • Kim, Mi-Young;Lee, Yu-Il
    • Clinical and Experimental Reproductive Medicine
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    • v.34 no.2
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    • pp.117-124
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    • 2007
  • Objective: The aim of this study was to compare the efficacy of slow freezing with vitrification method for cryopreservation of mouse pronuclear stage embryos. Methods: Mouse pronuclear embryos obtained from superovulated mice and classified into 2 groups of slow freezing and vitrification. Slow freezing solution consisted of 1.5 M PROH, 0.1 M sucrose, while vitrification solution consisted of 40% ethylene glycol, 18% Ficoll and 0.5 M sucrose diluted in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. Recovery and survival rates after thawing and development rates to hatching balstocyst stage were compared between two groups. Results: After freezing and thawing, recovery rate of slow freezing group was 93.8%, whereas vitrification group was 66.5% (p<0.01). Survival rate of recovered embryos were similar between two groups as 83.2% in slow freezing and 87.6% in vitrification. Embryo development rates to 2-cell stage after 24 hrs (77.0% vs 59.1%), 4-cell after 48 hrs (72.6% vs 53.3%), blastocyst after 96 hrs (53.1% vs 40.1%) of thawing were significantly higher in vitrification group than those of slow freezing group, respectively. Conclusion: The vitrification method may provide better developmental competence of frozen-thawed embryos than that of slow freezing method for cryopreservation of mouse pronuclear stage embryos.

A Simple Embryonic Stem Cell-Based in vitro Differentiation System That Recapitulates Early Erythropoietic Events in the Mouse Embryo (생쥐 배아에서의 초기 적혈구 분화를 재현 할 수 있는 배아주 세포에 기초한 간단한 시험관내 분화체계)

  • 김철근
    • The Korean Journal of Zoology
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    • v.39 no.3
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    • pp.239-247
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    • 1996
  • An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.

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생쥐 초기 배아의 'In Vitro 2-Cell Block'현상에 관한 연구

  • 김해권;공희숙;조완규
    • The Korean Journal of Zoology
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    • v.29 no.1
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    • pp.13-22
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    • 1986
  • In order to investigate the 'In Vitro 2-Cell Block' phenomenon found in certain mouse strains such as ICR, the present studies have been done. Fertilized eggs (1-cell) and 2-cell embryos recovered from the oviducts of the ICR mouse at the various time intervals after hCG injection to induce ovulation were cultured for 3 or 4 days to examine the capability for further cleavage beyond 2-cell stage. Consequently, it was found that some proportions of the 1-cell or 2-cell embryos recovered at 30 hours post hCG showed their cleaving capability and if the embryos were obtained after 48 hours of hCG injection, they were all at 2-cell stage and most of them developed to the blastocysts in vitro. It was also found that the embryos obtained at 27 hours post hCG showed their stronger capacity of further development in the groups cultured for shorter period than 24 hours in vitro before transferring to the oviduct. Based on the results, it can be inferred that mouse fertilized eggs should be remained inside the oviduct for a certain length of period after fertilization, or they should be cultured for a short period than 12 hours before returning back to the oviduct in order to develop to blastocysts. It was also found that though the embryos under the 2-cell block in culture showed normal feature up to 24 hours under the microscopical observation, they had already lost their capacity for the normal development, and if the culture of the 2-cell embryos was extended to 48 hours, they showed nuclei with heteropyknosis, and the vacuoles were were detected in the cytoplasm of embryonic cell if they were cultured for 72 hours.

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Establishment of Embryonic Stem Cell Line from ICR Mouse Blastocyst (ICR 생쥐 배반포로부터 배아주세포계통 확립)

  • 박성은;변태호;김용철;김종수;이상호
    • Journal of Embryo Transfer
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    • v.9 no.3
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    • pp.213-220
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    • 1994
  • 생쥐 배반포로부터 내부세포괴(inner cell mass, ICM)를 outgrowth로 분리하여 증식 시킴으로써 배아주(embryonic stem, ES)세포를 확립하고자 본 실험을 실시하였다. 과배란처리와 교미에 의해 생산된 ICR 생쥐의 3.5일 배반포를 sDMEM내의 배아성 섬유아단흥배양층에 배양하여 ICM세포의 증식을 조사한 결과, 3.5일부터 분리한 ICM세포들은 배양 7, 8일에 각각 1,500 및 3,200세포의 미분화세포로 증식하였다. 이들 세포의 계대배양에 의해 잠정적인 ES세포 colony를 얻었으며 10회의 계대배양후에도 그 형태가 변하지 않았다. 이들 세포는 다능성의 분화능을 보여 전형적인 ES세포 형태를 보였다. 이 같은 결과는 ICR배반포에서 outgrowth로 분리한 ICM으로부터 ES세포 확립이 가능함을 보여준 것이다.

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Effect of Hydrosalpingeal Fluid on the Implantation in-vitro in a Murine Model (생쥐 배아의 체외배양 시 착상과정에 대한 난관수종액의 영향)

  • Jun, Jin-Hyun;Koong, Mi-Kyoung;Lim, Chun-Kyu;Kim, Soo-Kyung;Kang, Inn-Soo
    • Clinical and Experimental Reproductive Medicine
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    • v.27 no.2
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    • pp.159-164
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    • 2000
  • 연구목적:인간의 체외수정 및 배아이식술에서 난관수종을 갖는 환자에서 임신율과 착상률이 감소된다는 보고들이 있지만 이에 대한 명확한 기작은 밝혀지지 않았다. 본 연구에서는 생쥐 배아를 이용한 체외 착상모델에서 인간의 난관수종액(HSF)이 착상과정에 미치는 영향을 알아보고자 하였다. 연구재료 및 방법 난관수종액은 난관수종으로 수술을 받은 8명의 환자로부터 채취하였으며, 실험에 사용하기 전까지 냉동고에 보관하였다. 생쥐의 포배기 배아는 2-세포기배아를 3일 동안 배양하여 그 중 상태가 양호한 포배기 배아만을 선별하여 투명대를 제거한 후 사용하였다. 기본 배양액으로는 Ham's F-10을 사용하였으며, 배양 시 기본 배양액만을 사용한 경우를 group Ⅰ으로 하였고, 기본 배양액에 0.5% FBS를 첨가한 경우를 group Ⅱ, 0.5% FBS와 50% HSF를 첨가한 경우를 group Ⅲ , 100% HSF에 0.5% FBS를 첨가한 경우를 group Ⅳ,100% HSF만을 사용한 경우를 group Ⅴ로 하였다. 투명대를 제거한 포배기 배아를 각각의 HSF에 대한 5종류의 배양액에서 48시간 동안 배양하였다. 체외 착상 유무는 부착 부위에서 크기가 커진 영양세포들을 관찰하여 판정하였으며, 착상 부위의 표면적은 화상분석기를 이용하여 산출하였다. 결 과: 생쥐 배아의 체외 착상률은 group Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ에서 각각 0%, 98.9%, 77.5%, 40.4%, 10.0%로 나타났으며, 착상 부위의 평균 표면적은 group Ⅱ, Ⅲ, Ⅳ, Ⅴ에서 각각 $74,675{\pm}25,201{\mu}m^2$, $59,024{\pm}25,877{\mu}m^2$, $45,156{\pm}22,654{\mu}m^2$, $38,254{\pm}17,115{\mu}m^2$이었다. 체외 착상률과 부위의 표면적은 HSF의 농도가 증가함에 따라 통계적으로 유의하게 감소하였다(p<0.001). 결론:인간의 난관수종액(HSF)은 생쥐 배아의 체외 착상과 영양배엽세포의 증식을 억제하는 것으로 확인되었으며, 이러한 원인이 난관수종을 갖는 환자에서 임신율이 낮은 것과 밀접한 관련이 있을 것으로 생각된다.

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