摘要
Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.
Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency.
作者
AN Li-long, YANG Qi, XIAO Mei, FENG Xiu-Liang, YANG Chun-rong, LEI An-min, GAO Zhi-min, DOU Zhong-ying and QIU Huai( Agriculture College of Zhanjiang Ocean University, Zhanjiang 524008 , P. R . China
Key laboratory of AnimalReproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China , Northwestern Scienceand Technology University of Agriculture and Forestry, yangling 712100 , P.R. China)
基金
supported by the National Natural Science Foundation(C36970359).
