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Factors Influencing the Isolation and Culture of Mouse Embryonic Stem Cells and Bovine Embryonic Germ Cells
Author: XuXiaoMing
Tutor: ZuoZhongYing
School: Northwest University of Science and Technology
Course: Clinical Veterinary Medicine
Keywords: BALB / c mice Cow Embryonic stem cells Embryonic germ cells Primordial germ cells Immunosurgery
CLC: S814.8
Type: Master's thesis
Year: 2003
Downloads: 145
Quote: 5
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Abstract
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BALB / c mice, the research materials, establish immune surgical separation cloned mouse ES cells; and bovine fetal material isolated from bovine primordial germ cells cloned bovine EG cells. Bovine EG cells isolation and cloning of mouse ES cells, influencing factors are discussed, and laid the foundation for the establishment of mouse ES cell lines and cattle EG. The experimental results are as follows: 1. The by immunosurgery isolated from 45 BALB / c mouse blastocyst ICM, 20 removal of trophoblast cells seeded in MEF feeder layer, using DMEM (high glucose) with 15% FBS 0.1mMβ-mercapto ethanol 0.01mM non essential amino acids 1000 IU / ml LIF 100 IU / mL penicillin 100 IU / ml streptomycin culture medium, 17 to form the typical ICM colony, an embryo passaged for 10 generations. For the whole embryo culture BALB / c mouse embryo 102, use same immunosurgery, culture method, 66 a typical ICM colony, in which an embryo spread to the 8th generation. 2. Collect pregnancy 29 ~ 100d the bovine fetal 46 cases, with DMEM (high glucose) 15% NBS (or FBS) 0.1mMβ-mercapto the 0.01mM non-essential amino acids cattle ethanol 2mM glutamine bucket 100 IU / mL penicillin 100 IU / ml streptavidin hormone culture medium separation of cloned cattle EG cells or analogs of primordial germ cells from genital ridge (glandular), 25 cases of EG cell colony, the highest one case spread to the 6th generation. Gestational age 29 ~ 45d cow fetus is most suitable for the isolation and cloning of bovine EG cells, gestational age greater than 70 days, and is difficult to get the cattle EG cells. 3. Isolated and cultured from 45d bovine fetal bovine fetal fibroblast cells, spread to 28 generation. Α-MEM is a good cow fetal fibroblasts were isolated and cultured medium. 4. Adherence rate of mouse embryos and ICM sets colony formation rate in the MEF, STO and BEF feeder layer was no significant difference (P> 0.05); MEF, STO, BEF and HEF feeder layers could promote the proliferation of bovine EG cells in vitro, but STO the best, the the MEF and the BEF followed by, HEF worst effect. 5. DMEM medium were isolated and cultured and passaged bovine PGCs significantly better than the F12; 15% NBS adherence rate in mouse embryonic and ICM set off the formation rate was significantly better than NBS 10% (P <0.05); than 15% of the 15% FBS. NBS is conducive to mouse ES cells (P <0.05), the isolation and cloning of bovine EG cells. 6. Add LIF (10ng/ml) favor mouse ES cells (P <0.05), the isolation and cloning of bovine EG cells; LIF, SCF and bFGF (Add concentration was 10 ng / ml) can significantly promote the three factors combined effects clone cattle EG cell separation. 7. BALB / c mouse ES cells to pancreatin is very sensitive, 0.05% trypsin and 0.008% EDTA ES cells are preferred digestive on cell injury force is small, and after passage ES cell colony forming ability was also higher (P < 0.05). Isolated mouse ES cells, bovine EG cells, morphological observation, the AKP (PAS) staining, in vitro differentiation experiments karyotype analysis that has many of the characteristics of embryonic stem cells.
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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > General Animal Science > Livestock and poultry breeding > Reproductive biotechnology
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