【摘要】 本文主要探讨了不同精子处理对应用显微授精介导(intracytoplasmic sperm injection-mediated trans-genesis,ICSI-Tr)生产转基因水牛胚胎效率的影响。本试验以p18T-BCN5.2-IFN-1.1pA-EGFP为载体,比较了精子的不同处理方式(NaOH和冻融)、NaOH处理精子不同时间(5min,10min,20min,30min和60min)以及不同外源DNA浓度(5ng/μL,10ng/μL,25ng/μL和50ng/μL)对ICSI-Tr转基因效率的影响。结果显示:NaOH处理组的囊胚EGFP表达率(46.1%),与冻融精子处理组(47.1%)差异不显著,但发育率显著高于冻融处理精子组(28.3%vs16.7%,P<0.05)。处理60min组和30min组的分裂率分别为78.9%和77.5%,显著高于20min、10min和5min组的64.0%、60.0%和64.0%(P<0.05),其中30min处理组的囊胚EGFP表达率高达44.4%,显著高于其它处理组(P<0.05)。25ng/μL组和50ng/μL组的早期胚胎EGFP表达效率差异不显著(41.3%vs42.5%),但显著高于5ng/μL组和10ng/μL组(22.5%和35.5%),25ng/μL组的囊胚EGFP表达效率显著高于其它组(P<0.05)。本研究优化了应用ICSI-Tr生产转基因水牛胚胎技术体系,为获得ICSI-Tr转基因水牛奠定了良好的工作基础。更多还原

【Abstract】 Effects of sperm treatment on efficiency of production transgenic buffalo embryo using intracyto-plasmic sperm injection-mediated transgenesis (ICSI-Tr) was explored in the present study.With p18T-BCN5.2-IFN-1.1pA-EGFP as transgenic vector,effects of sperm treating methods (NaOH or freeze-thawing),sperm treating time with 10 mmol/L NaOH (5 min,10 min,20 min,30 min and 60 min) and concentration of exogenous DNA with sperm (5 ng/μL,10 ng/μL,25 ng/μL and 50 ng/μL) on transgenic efficiency were investigated.The results showed that:In NaOH treating group 46.1% blastocyst expressing EGFP,similar to that of freeze-thawing group (47.1%),but embryos developed to the blastocyst stage was significantly higher than that of freeze-thawing group (28.3% vs 16.7%,P<0.05).The zygotes cleavage rates were 78.9% and 77.5% respectively,when sperm was treated 60 min and 30 min,which were significantly higher than that 5 min,10 min and 20 min treatment groups (64.0%,60.0% and 64.0%,P<0.05).In 30 min NaOH treatment group 44.4% blastocyst expressing EGFP,which was significantly higher than that of other groups.With different DNA concengtration treatment,EGFP expressing rate of early embryos (41.3% vs 42.5%) in 25 ng/μL and 50 ng/μL groups were significantly higher than that of 10 ng/μL and 5 ng/μL groups (22.5% and 35.5%),and the rate of blastocyst expressing EGFP in 25 ng/μL group was significantly higher than that of other groups.This study optimized the process of production transgenic buffalo embryo using ICSI-Tr technique,which would do help in production transgenic buffalo in the future.更多还原