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ID 7811
Eprint ID
7811
フルテキストURL
Thumnail O002338.pdf 502 KB
タイトル(別表記)
Studies on culture and application of embryonic stem cells in the mouse
著者
徳永 智之 岡山大学
抄録
Embryonic stem (ES) cells isolated from mouse preimplantation embryos can be maintained as a pluripotent cell line in culture. Since ES cells are capable of contributing to a wide variety tissues including the germ line, when they are injected into host embryos, they are considered to be the useful tool for the production of transgenic mice. However, these abilities of ES cells tend to lose during cul ture in long term, or in the process in transfection with foreign gene and selection process. The purposes of the present study is 1) to isolate new ES cell lines, 2) to examine the culture conditions for maintenance of them, 3) to develop efficient method for production of viable germ line chimaeras and 4) to demonstrate the production of transgenic mice. I. Studies on the isolation of embryonic stem cell lines from mouse blastocysts and inner cell masses in the mouse The mouse blastocysts or inner cell masses (ICMs)isolated were cultured on feeder cell layer of the primary embryonal fibroblast cells inactivated with mitomycin C treatment. Totally, 11 ES cell lines were isolated from 2l0 blastocysts or ICMs obtained from sevral different mouse strains. There was no difference in the efficiency of the ES cell line isolation among the mouse strains and between blastocysts and ICMs. Colonies of these cells showed formed EC cell like morphology forming compact islands of cells wi th unclear cell borders. Three of ES cell lines, TT-12, TT-B4 and F1/1, had diploid chromosomes with the proportions of 78%, 78% and 72% respectively. In order to determine the differentiation potential of the ES cells in vitro TT-12 and F1/1 cells were cultured in suspentlon without feeder cell layer. They differentiated spontaneously into the cystic embryoid bodies which contained variety of tissues including endoderm and ectoderm like cells, indicating pluripotency of the cells in vitro. II. Studies on the culture conditions to maintain embryonic stem cells. The proligeration activity and undifferentiation conditon of F1/1 cells, which were isolated from a blastocyst obtained from C57BL/6 female mated with CBA male, were examined when they were cultured with different feeder cell layers or with Leukemia inhibitory factor (LIF). The proportions od colony formation and colonies with positive for alkaline phosphatase were higher when F1/1 cells were cultured for 3 to 7 days with primary embryonal fibroblast cells than with the STO cells. These feeder cell layers are indispensable to inhibit the differentiation of F1/1 cells during culture. When F1/1 cells were cultured through 15 passages with LIF without feeder cell layer, they were maintained undifferentiated and 68% of them had normal karyotype (38+XY). However, no young were obtained when chimaeric embryos produced between 8 cell embryos and F1/1 cells maintained with LIF were trasferred to the recipients, while 50 to 88% of young obtained from chimaeric embyos with F1/1 cells maintained in culture with feeder cells were chimaeras. Although many ES cell lines have been isolated from several mouse strains, the cell lines to produce germ line chimaeras have been limited to the l29 strain origin. The present study demonstrated that F1/1 cells isolated from a blastocyst having the C57BL/6xCBA genotype could produce germ line chimaeras by injecting the cells into blastocysts and 8-cell embryos. The chimaera production rate using CD-1 blastocysts as the host was wery low (20%) as reported by other investigaters. However, when F1/1 cells were introduced into the perivitteline space of 8-cell embryos obtained from CD-1 strain mice, very high proportion (80%) of chimaeric mice be produced and the chimaeric mice showed extremely high chimaerism. In the breeding tests, germ line chimaeras were indicated in 89% of fertile male and most of them had spermatozoa derived only from F1/1 cells. No live youg were obtained when chimaeric eggs, which were produced by injecting F1/1 cells into parthenogenetic 8-cell eggs, were transferred. IV. Studies on the production of transgenic mice using ES cells F1/1 cells transformed with the neomycin phosphotransferase (neo) gene using electroporation methods were selected for G4l8 resistance and tested for its ability to produce chimaeric mice. Two types of wave forms, square wave and exponential decay, were used for electroporation. There was no difference between square wave and exponential decay in the efficiency of transformation of F1/1 cells. Southern blot hybridization analysis demonstrated that four of neomycin resistant ES cell lines had neo gene sequences in their genomic DNA but another two cell lines lacked it. The karyotype of them appeared to be stable, as it had still euploid chromosome constitution after transformation and the selection. Two of the lines contained significant populations of the cells with chromosomal abnomality. Overt coat color chimaeras were obtained and their germ lines were transmitted with all four neo resistant ES cell lines tested. Southern blot hybridization and PCR analysis of genomic DNA confirmed again that F(1) and F(2) generation of these germ line chimaeras were transmitted with neo gene stably. These results indicate that ES cells (F1/1) isolated from blastocyst of C57BL/6 female mated wi th a CBA male can be used as a vehicle for transgenesis.
発行日
1991-09-30
出版物タイトル
資料タイプ
学位論文
学位授与番号
乙第2338号
学位授与年月日
1991-09-30
学位・専攻分野
博士(農学)
授与大学
岡山大学
学位論文本文
学位論文(フルテキストURL参照)
言語
Japanese
OAI-PMH Set
岡山大学
論文のバージョン
publisher
査読
不明