小鼠无透明带胚胎培养方法的优化  

Optimization of Culture Method for the Zona-free Embryos of Mouse

作　　者：刘根胜[1] 刘军[1] 孙健红[1] 刘凤军[1] 张东[1] 刘利杰[1] 马夜肥[1] 杨鹭[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,杨凌712100

出　　处：《农业生物技术学报》2008年第2期270-275,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2001AA213081)资助

摘　　要：探索一种高效的无透明带胚胎培养方法,为手工核移植提供技术支持,研究以小鼠(Mus musculus)胚胎为材料,对比了现有的3种无透明带胚胎培养方法,对其中效果较好的WOW(the well of the well)法进行改进,以海藻酸钙凝胶包埋小凹,并且尝试与共培养技术相结合。结果显示,海藻酸钙凝胶包埋小凹法(AEW法)的囊胚发育率(54.7%)和囊胚细胞平均数(54.3)显著高于微滴单卵培养法(MDS法)和琼脂柱包埋培养法(AE法)(P<0.05),与WOW法相当(P>0.05);使用AEW法进行培养时,最佳的海藻酸钠和钙离子浓度分别为0.7%和1.5%;该法与颗粒细胞共培养相结合后,胚胎卵裂率(89.7%)与囊胚发育率(67.9%)显著高于对照组(分别为79.5%和54.7%)(P<0.05)。实验结果表明,AEW法培养无透明带胚胎的效果优于其它几种方法;AEW法与颗粒细胞共培养相结合可以大大提高无透明带胚胎的胚胎卵裂率及囊胚发育率。To search for an effective culture method for zona-free embryos and provide technical support for hand made cloning, this study contrasted three culture methods for the zona-free embryos of mouse（Mus musculus）, modified the well of the well （WOW） culture method by embedding the well with calcium alginate gel and tried to combine with the co-culture technology. The results showed that the blastocyst development rate （54.7%） and the average cell numbers of blastocysts （54.3） culturing with the calcium alginate gel embedding well（AEW） culture method were significantly higher than those of culturing with the micro-drops single（MDS） culture method and agarose-pillar embedding （AE） culture method （P 〈0.05）, but no significant differences （P 〉0.05） with the WOW culture method（P 〉0.05）; the optimal concentrations of the sodium alginate and calcium ion were 0.7% and 1.5% respectively; the cleavage rate （89.7 %） and the blastocyst development rate （67.9%） culturing with the AEW culture method combining with co-culture technology were significantly higher than those of culturing with the AEW culture method only （P 〈0.05）. The results indicate that the efficiency of AEW culture method is better than that of the other culture methods, combining with granule cells co-culture technology can greatly improve the cleavage rate and the blastocyst development rate in culturing zona-free embryos.

关 键 词：小鼠 无透明带胚胎培养 海藻酸钙凝胶包埋小凹法 

分 类 号：S185[农业科学—农业基础科学]