CAJ | 학술논문

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。
본문주요탐토료불동정자처리대응용현미수정개도（intracytoplasmic sperm injection-mediatedtrans-genesis，ICSI-Tr）생산전기인수우배태효솔적영향。본시험이p18T—BCN5．2-IFN-1．1pA—EGFP위재체，비교료정자적불동처리방식（NaOH화동융）、NaOH처리정자불동시간（5min，10min，20min，30min화60minl이급불동외원DNA농도（5ng／μL，10ng／μL，25ng／μL화50ng／μL）대ICSI-Tr전기인효솔적영향。결과현시：NaOH처리조적낭배EGFP표체솔（46．1％），여동융정자처리조（47．1％1차이불현저，단발육솔현저고우동융처리정자조（28．3％VS16．7％，P〈0．05）。처리60min조화30min조적분렬솔분별위78．9％화77．5％，현저고우20min、10min화5min조적64．0％、60．0％화64．0％（P〈0．05），기중30min처리조적낭배EGFP표체솔고체44．4％，현저고우기타처리조（P〈0．05）。25ng／μL조화50ng／μL조적조기배태EGFP표체효솔차이불현저（41．3％VS42．5％），단현저고우5ng／두조화10ng／μL조（22．5％화35．5％），25ng／μL조적낭배EGFP표체효솔현저고우기타조（P〈0．05）。본연구우화료응용ICSI-Tr생산전기인수우배태기술체계，위획득ICSI-Tr전기인수우전정료량호적공작기출。
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.