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1、农业生物技术学报JournalofAgriculturalBiotechnology2007,15(2):212~216·研究论文·小鼠卵母细胞去核方案的优化*刘军,王勇胜,刘凤军,李学家,张涌**(西北农林科技大学生物工程研究所，杨凌712100)摘要：研究对比了透明带磨口刺入和透明带直接刺入两种去核方法、蔗糖处理、细胞松弛素B(CB)浓度去核针内径对小鼠卵母细胞去核效率，及CB浓度对去除部分胞质的孤雌胚早期发育的影响。结果：(1)添加3％蔗糖与对照组之间的去核效率无影响(>0.05)；(2)CB浓度为5、10和20滋g/mL时，磨口法的去核成功率差异不显著(>0.05)，刺入法
2、在CB浓度为5和10滋g/mL时去核成功率显著高于20滋g/mL组(<0.05)，磨口法去核成功率显著高于刺入法(<0.05)；(3)去除部分胞质的孤雌胚卵裂率各实验组之间差异不显著(>0.05)，20滋g/mLCB处理组囊胚率显著低于其它处理组(<0.05)；(4)在去核针内径为10、15和20滋m时，磨口法去核成功率差异不显著(>0.05)，刺入法应用20滋m去核针的去核成功率显著低于其它组(<0.05)。表明，磨口法在10滋g/mLCB和15滋m内径去核针条件下提高了去核效率(成功率为90.8%，速度为50s/个)，且不影响孤雌胚的体外发育率。关键词：小鼠；卵母细胞；去核方法
3、；蔗糖处理；细胞松弛素B；去核针中图分类号:S185文献标识码:A文章编号:10061304(2007)02021205OptimizationoftheEnucleationProtocolsforMouseOocytesLIUJun,WANGYongsheng,LIUFengjun,LIXuejia,ZHANGYong**Tooptimizetheenucleationprotocolsofmouseoocytes,theeffectsoftheenucleationmethodsofabradedingthezonapellucida(AZP)orstickingthezon
4、apellucida(SZP)straightly,sucrosetreatment,concentrationsofcytochalasinB(CB)andinnerdiameterofpipettesonenucleationefficiency，andtheeffectsoftheconcentrationofCBonparthenogeneticembryosremovedpartialcytoplasmfrom1/12to1/8wereexamined.Theresultsshowedthat,(1)therewerenosignificantlydifferencesb
5、etweensupplemented3%sucrosegroupandcontrolgrouponmanipulatingtimeandenucleationrateachievedbyAZPorSZP(＞0.05);(2)5,10and20滋g/mLCBhadnosignificanteffectsonmanipulatingtime(＞0.05).AZPhadnosignificantdifferentenucleationratesamong5,10and20滋g/mLCBgroups(＞0.05)；however,inSZP,theenucleationratesof5an
6、d10滋g/mLCBweresignificantlyhigherthanthatof20滋g/mLCB(<0.05);(3)thecleavagerateofparthenogeneticembryoshadnosignificantdifferencebetweenvariousgroups(＞0.05).Blastocystrateof20滋g/mLCBgroupwassignificantlylowerthanthatofothergroups(<0.05);(4)theenucleationratesof10,15and20滋minnerdiameterofpipette
7、werenosignificantlydifferentinAZP(＞0.05);theenucleationratesof10and15滋minnerdiameterofpipetteswerehigherthanthatof20滋minSZP(<0.05).TheenucleationratesachievedbyAZPwassignificantlyhigherthanthatachievedbySZP(<0.05).Theresultsindicatethat
