| 不同精子处理对应用ICSI—Tr技术生产转基因水牛胚胎效率的影响 |
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| 引用本文： | 刘庆友,王丹,张会娜,陈自洪,孟凡丽,陆凤花,石德顺.不同精子处理对应用ICSI—Tr技术生产转基因水牛胚胎效率的影响[J].广西农业生物科学,2011,30(4):294-299. |
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| 作者姓名： | 刘庆友 王丹 张会娜 陈自洪 孟凡丽 陆凤花 石德顺 |
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| 作者单位： | 广西大学亚热带生物资源保护利用国家重点实验室,南宁,530004 |
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| 基金项目： | 国家转基因重大专项，国家自然基金，广西自然基金，南宁市科技攻关 |
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| 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-mediatedtrans-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60minl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％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／肚组和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。
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| 关 键 词： | 水牛 ICSI—Tr 转基因胚胎 |
Effects of Sperm Treatment on Efficiency of Production Transgenic Buffalo Embryo Using ICSI-Tr Technique |
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| Liu Qingyou Wang Dan Zhang Huina Chen Zihong Meng Fanli Lu Fenghua Shi Deshun.Effects of Sperm Treatment on Efficiency of Production Transgenic Buffalo Embryo Using ICSI-Tr Technique[J].Journal of Guangxi Agricultural and Biological Science,2011,30(4):294-299. |
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| Authors: | Liu Qingyou Wang Dan Zhang Huina Chen Zihong Meng Fanli Lu Fenghua Shi Deshun |
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| Institution: | Liu Qingyou Wang Dan Zhang Huina Chen Zihong Meng Fanli Lu Fenghua Shi Deshun( State Key Laboratory for Conservation and Utilization of Agricultural Bioresources in the Subtropics, Guangxi University, Nanning, 530004) |
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| 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. lpA-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 fi＇eeze-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 rain 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 transgenie buffalo embryo using ICSI-Tr technique, which would do help in production transgenic buffalo in the future. |
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| Keywords: | Buffalo Intracytoplasmic sperm injection-mediated transgenesis （ICSI-Tr） Transgenic embryo |
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