The Isolation And Cloning Of Sheep's Embryonic Stem-Like Cells

Studies the sheep like-ES cells in vitro,not only as an model of mammalian development in vitro,but also can be used to treat and reproduction,a bright future at prospect of application. At present,the research of embryonic stem cell are focused on purification,amplification,and directional induced differentiation,low efficiency of establishing mammalian ES cell lines is still exist,and many other problems remain unsolved,Even,successful establishment is still not reported in today. In this study,we were isolate and culture ESCs from sheep embryos in different development stages,by adopting the methods of mouse and human,and to compare some influencing factors that the efficiency of establishing about stem cell lines. It were Provided a reference which in order to as further development of sheep ES cell lines. The results obtained were as follows:1,Using 93 sheep embryos in experiment, 80 completion of incubation, ICM formation rate was 48.4%; a total of 35 ES-cell mass were formed,the formation of ES cell group was 37.6%.Test MEF,SEF feeder to isolated and cultured ES-cells in sheep. Final results: Two kinds of feeder cells can promote promote the proliferation of ES cell sheep,and their rate of hatching were 71.4% (5 / 7),66.7% (8 / 12); rate of ICM proliferation were57.1% (4 / 7 ),41.7% (5 / 12),were not significantly different,and wo found that the proliferation of ICM were slowly and limited in the SEF,therefore, MEF are more suitable for sheep ES cells .2,Rate of attachment and ICM proliferation are varied at different stages of embryos in sheep .Rate of adherent blastocyst (75%) and rate of adherent dense morula (62.5%) is relatively high;rate of the proliferation rate of blastocyst ICM (47.9%),compact morula ICM proliferation (57.1%),than rate of morula ICM proliferation (25.0%) and rate of hatching blastocyst ICM proliferation (44.4%). Under the microscope,the same stage embryos,rate of attachment and proliferation in embryo,A-class is higher than the B-class. The results showed: compact morula and blastocyst is more suitable for sheep ES-cell isolation and culture.3,Through the experimental analysis,wo know : alone or in combination FBS,KSR and ESCCM,separation and passage on sheep like- ES cell. they can promote the growth of ES-cells,which of (KnockoutTMDMEM/F-12 +5% FBS +15% KSR + LIF (1000 IU / mL) and bFGF (20 ng / mL))culture system,it were discreted cell clumps by mechanical passage method,the maximum is 8 generations which spread to sheep ES. And under the same conditions,(5% FBS +15% KSR +20% ESCCM )training system can also be transmitted to 7 generations.4,The experiment was made (KnockoutTMDMEM/F-12 +5% FBS +5% KSR +0.1 mmol / Lβ-Me +0.1 mmol / L NEA (non-essential amino acid) +2 mmol / L glutamine +2 mmol / L pyruvate sodium +100 IU / mL of penicillin +100μg / mL streptomycin +1000 IU / mL LIF + (20 ng / mL) bFGF) based culture medium. added 20,40,60 ng / mL of three different SCF,was found that its no significant effectto promote ES-cell clone .5,This experiment also research that the frozen were used to isolation and culture the embryo ES cell,The results obtained, as long as operating normal the freezing process, resurrection normal of embryos,they can hatch,ICM proliferation and passage.6,The isolation and culture sheep like- ES-cell in vitro,identified by morphological observation,AKP staining,karyotype analysis,Nanog testing,differentiation experiments in vitro,and found that ES-cell:typical morphology,strong positive performance in AKP,showing a normal diploid karyotype,and can express pluripotent cell factor Nanog,gained embryoid bodies,so it demonstrated that they have many characteristics about embryonic stem cells.