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대한생식의학회지 제27권 제1호 2010년
착상 전 유전진단 기술 개발의 동물실험 모델로서 할구 생검된 생쥐 배아에서 동결보전 융해 후 배아 발생 양상과 공배양 효과에 관한 연구
서울대학교 의과대학 산부인과학교실1, 의학연구원 인구의학연구소2, 한국과학기술원 의과학연구센터3, 울산대학교 의과대학 산부인과학교실4
김석현1,2, 김희선2, 류범용2, 최성미2, 방명길3, 오선경2, 지병철1,2, 서창석1,2, 최영민1,2, 김정구1, 문신용1,2, 이진용1, 채희동4, 김정훈4,
Developmental Competence and Effects of Coculture after Cryopreseravation of Blastomere-Biopsied Mouse Embryos as a Preclinical Model for Preimplantation Genetic Diagnosis
Seok Hyun Kim1,2, Hee Sun Kim2, Buom Yong Ryu2, Sung Mi Choi2, Myung Geol Pang3, Sun Kyung Oh2, Byung Chul Jee1,2, Chang Suk Suh1,2, Yong Min Choi1,2, Jung Gu Kim1, Shin Yong Moon1,2, Jin Yong Lee1, Hee Dong Chae4, Chung Hoon Kim4
Department of Obstetrics and Gynecology, College of Medicine1, Institute of Reproductive Medicine and Population, Medical Research Center2, Soul National University, Biomedical Research Center, Korea Advanced Institute of Science and Technology3, Department of Obstetrics and Gynecology, College of Medicine, Ulsan University4, Seoul, Korea
Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL♀/CBA♂). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode’s solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethy1 sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomerc-biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham’s F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond lastocyst stage were stained with 100% Giemsa to count the total number of nuclei in each embryo. Results: The surviral rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the blastomere-biopsied groups, but it was significantly lower than in the non_cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group (50.2±14.0) than in 6/8 (26.5±6.2), 5/8 (25.0±5.5), and 4/8 (17.8±7.8) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group (25.9±10.2), compared with the control (50.2±14.0), 7/8 (56.0±22.2), and 6/8 (55.3±25.5) groups. Conclusion: After cryopreservation, Blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.
키워드 : Preimplanation genetic diagnosis (PGD), Mouse embryo, Blastomere biopsy, Cryopreservation, Cocuture, Vero call, Blastocyst
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