Isolation And Clone Of Bovine Embryonic Stem Cells

Embryonic stem (ES) cells are pluripotential stem cells derived from animal embryos or primordial germ cells (PGCs) . ES cells can be cultured in vitro and kept undifferentiated. After being reintroduced into embryos, ES cells will involve in the development of tissure and organs of recipient embryos and can be transmitted through the germ line. So,foreign genes can be introduced into animal genome with ES cells, transgenic animal and clone animal can be produced by ES cells. Moreover,the most important application of ES cells is to produce recessive mutation, to understand gene functions by targeted mutagensis, to cre-at animal modle of human genetic disease and to understand the law of mammalian animal development. The object of this research were to bulid the technical system on isolation and clone of bovine ES cells. The main contents were as follows :1. Building the technical system on isolation and clone of murine embryonic and bovine testicular fibroblasts. The conclusions are as follow: â‘  15 passage murine embryonic fribroblasts (MEF) and 5 passage new bovine testicular fibroblasts (NBTF) were isolated and cloned in media (1.0g/L glucose DMEM (Gibco) supplemented with 15% NBS, 0. 1mM β-mercaptoethanol(Sigma), 0. 1μM Na₂SeO₃. The murine embryonic and new bovine testicular tissures were cut and incubated at 37℃ in 0. 25% trypsin / 0. 04% EDTA; â‘¡ Compared with MEF, the length and width of NBTF are big, inddition to, NBTF cell grow fast; â‘¢ Murine embryos from 12d to 16d were most suitable for isolationand clone of MEF; (4) Treated with 0. 25%trypsin + 0. 04% EDTA, single MEF cell and single NBTF cell were isolated and cloned by short term (from 2min to 3min)j (5) To supplement with 0. l\iM Na2SeO3, 15% NBS and 0. 1 mM (3-mercaptoethanol is beneficial to reproduction of MEF and NBTF cells.2. Studies on cryopreservation of NBTF and MEFMethords for cryopreservation of MEF and NBTF were investigated in this paper. The results showed as follow: ? The vitality of MEF cells and NBTF cells exposed to the freezing media n (PBS supplemented 0. 4% BSA, 0. 1M sucrose* 1. 5M ethylene glycol(EG)) higher than freezing media I (DMEM + 10% NBS + 10%DMSO). (2) When NBTF and MEF were freezed in media I , the balanced time can be reduced from 2. 5h to 0. 5h without influence on their vitality. Moreover, freezing rate can be increased from 0. 6°C/min to 1. 2°C/ min, induced ice did not influence on their vitality. On the vitality of NBTF cells and MEF cells, thawing media I PBS (supplemented 0. 1M sucrose, 0. 4% BSA)>thawing media I (DMEM) (supplemented 10% NBS)>thawing media M PBSA .3. Growth behavior of bovine embryos ICM on MEF feeder layerThe growth behavior difference between bovine embryos and murine embryos were studied on MEF feed layer. The results showed as follows: (T) After having spread, bovine embryonic trophoblast formed a transparent and membranous structure coving inner cell mass (ICM), however, murine embryonic trophoblast formed disc structure} (2) Bovine ICM formed four kinds of colonies with different morphology, including the mass-like, the net-like, the stream-like and the mixtured colonies; ? Compared with murine ICM, the bovine ICM grew more fast, so, at approximately day 3 to day 6 after plating for bovine embryos and day 3 to day 4 after plating for murine embryos, growing ICM were passaged at first j (4) The mixed colonies differented very early, while the others differentiated very late. Most like ICM grew in a defined spot, but stream-like and net like ICM proliferated fast and spread quickly. The single blastomeres from bovine late morula and murine late morula formed subblastophere. When the trophoblast were removed,ICM cell would differentiation rapidly.4. Isolation and clone of bovine ES cellsA total of 30 bovine morula were cultured in tissure medium (DMEM supplemented with 15% NBS, 0. lfiM Na2SeO3, 0. lmM 3 mercaptoethanol(Sigma) on a feeder layer of MEF or NBTF 5 days or 6 days later. The bovine ICM cells were disaggregated by a short-term trypsin treatment. The results sowed as follows: (T) Disaggregation of the ICM : ICM-derived cell embryos were selected following a 5-day to 6-day culture period and disaggregated with trypsin, then the smaller cellular fregment were transferred into a fresh feed layer, formation of 4 type of colonies, including ES-like cell colonies, fibroblast-like cell colonies, epithelioid like cell colonies and trophoblast like cell colonies;(2) Most of colonies derived mass ICM were ES-cells line consisted. However, colonies derived stream ICM were consisted of ES like cells and epitheliod like cells, most of colonies derived net ICM were composed of epithelioid like cells;(3) Compared with mouse ES cells, bovine ES cell grow slowly, so, ES cell were passaged at intervals of 4-days to 5-days. In addition to, bovine ES cell darker than murine ES cell. (4) Supplemented with 15% supper NBS (the ration of mouse embryo hatched are more than 86% within 24h culture); (5) Compared with the ration of ES colonies among TCM 199, DMEM (high glucose) and DMEM (low glucose), the ration of embryo hatched of DMEM (L) and DMEM (H) more than TCM 199. as result, The ration of ES colonies cultured in DMEM (L) are more than those cultured in DMEM (H) and in TCM199; (6) Super bovine embryos formed mass ICM, the ration of ES colonies are higher than stream ICM and net ICM derived normal embryos; (7) The ration of clone of ES cell were most high in DMEM (low glucose) culture medium supplemented with 15% NBS, 0. l|iM Na2Se03, 0.1 mM 3-mercaptoethanol, lOOOlU/ml LER and lOng/ml IGF; (8) ICM or ES colonies treated with 0. 05% trypsin and 0. 01% EDTA long term(6min), the ration clone of bovine ES cell were most high (75%); (9) 6 passage bovine ES like cell and 9 passage ES like cell were obtained in this research.