学辅助去核法对延边黄牛卵母细胞去核率及其重构胚发育率的影响.doc

化学辅助去核法对延边黄牛卵母细胞去核率及其重构胚发育率的影响 江西农业大学2011,33(2):0340—0344 ActaAgricuhuraeUniversitatisJiangxiensis E—mail:ndxb7775@sina.con 化学辅助去核法对延边黄牛卵母细胞去核率 及其重构胚发育率的影响 王玉涵,朱玉霞,尹多,李钟淑,方南洙 (延边大学农学院,吉林延吉133000) 摘要:为提高延边黄牛体细胞克隆的效率,采用化学辅助去核法——化学诱导剂脱羰秋水仙碱(Demecolcine, 简称Deme)处理体外成熟的M期延边黄牛卵母细胞,经显微操作构建重构胚后观察发育情况.试验研究了 Deme的处理浓度,作用时间对延边黄牛卵母细胞去核效果及其重构胚发育的影响.结果显示:体外成熟的卵 母细胞在0.2mL,0.3Ixg/mL,0.4g/mL的Deme溶液中处理45min,显核率与去核率无显着差异,去核 率达100%,但是0.2mL处理组的囊胚发育率显着高于其他处理组;45rain处理组的显核率显着高于 30nfin,60nfin处理组,45min处理组的囊胚率高于其他处理组;化学辅助去核法的囊胚率显着高于盲吸法. 化学辅助去核能够准确定位去除细胞核,并提高了延边黄牛体细胞克隆的去核率及重构胚的体外发育率. 关键词:延边黄牛;核移植;脱羰秋水仙碱;化学辅助去核 中图分类号:$823.812文献标志码:A文章编号:1000—2286(2011)02—0340—05 EffectsofChemicallyAssistedEnucleationontheEnucleationRate ofYanbianCattleOocytesandtheDevelopment oftheirRec0nstructedEmbryos WANGYu—han,ZHUYu—xia,YINDuo,LIZhong—shu,FANGNan—zhu (CollegeofAgriculture,YanbianUniversity,Yanji133000,China) Abstract:Inordertoenhancetheefficiencyofsomaticcellnucleartransfer,M11oocytesofYanbiancat. tleweretreatedbydemeeoleine.Nucleusofoocytesweredeletedbymicromanipulation.Theseoocyteswere preparedforthenucleartransferreceptor.Theeffectsoftheconcentrationofdemecolcine.theincubationtime ofoocytesindemecolcineontheenucleationratesandthedevelopmentofreconstructedemb~oswerestudied. Theresuhsshowedthat:Therewerenosignificantdifferencesintheratesofcytoplastprotrusionandenuclea. tioninvitromatureooeytesincubatedindemecolcineof0.2mL.0.3mL,0.4mLfor45rain. therateofcytoplastenucleationwas100%,the0.2g/mLgroupblastocystratewassignificantlyhigherthan thoseofotherconcentrations.Thegrouptreatedfor45minhadsignificantlyhighercytoplastprotrusionrate thanthegroupstreatedfor30minand60min.Thegrouptreatedfor45minhadhigherblastocystratethan othergroups.Theblastocystrateofchemicallyassistedenucleationwassignificantlyhigherthanthatofblinde- nucleation.Theseresultsdemonstratedthatthechemicallyassistedenucleationcanpreciselylocateand 收稿日期:2010—11—11修回日期:2011一O1—23 基金项目:国家自然科学基金项目和延边大学”211工程”三期建设项目 作者简介:王玉涵(1986一),男,硕士生,主要从事动