ICSI受精卵胞质注射生产转基因猪胚胎的初步研究

doi:10.16431/j.cnki.1671-7236.2018.06.024

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (6): 1618-1625.doi: 10.16431/j.cnki.1671-7236.2018.06.024

ICSI受精卵胞质注射生产转基因猪胚胎的初步研究

李红丽, 粟小平, 路粟雨, 刘帅, 刘庆友, 崔奎青, 石德顺

广西大学亚热带农业生物资源保护与利用国家重点实验室, 南宁 530000

收稿日期:2017-11-17 出版日期:2018-06-20 发布日期:2018-06-15
通讯作者: 崔奎青, 石德顺 E-mail:kqcui@126.com;ardsshi@gxu.edu.cn
作者简介:李红丽(1990-),女,河南周口人,博士,研究方向:动物生物技术,E-mail:939082246@qq.com
基金资助:
国家自然基金课题（31760648）；广西科研攻关项目（桂科攻1598013-2）；广西重点研发计划（桂科AB16380042）

Study on Transgenic Porcine Embryos by Cytoplasmic Injection into Zygotes Produced Using ICSI Method

LI Hongli, SU Xiaoping, LU Suyu, LIU Shuai, LIU Qingyou, CUI Kuiqing, SHI Deshun

State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi University, Nanning 530000, China

Received:2017-11-17 Online:2018-06-20 Published:2018-06-15

摘要/Abstract

摘要：

试验旨在探讨单精子胞浆内注射（intracytoplasmic sperm injection，ICSI）法生产猪体外受精卵和应用猪ICSI受精卵进行胞质注射生产转基因胚胎的可行性。首先对比了猪体外受精（in vitro fertilization，IVF）受精卵与ICSI受精卵的胚胎发育效率；然后观察了猪ICSI受精卵的双原核形成时间及效率，对精子注射到胞质后6~18 h分6个时间段进行地衣红染色，对比精子进入卵胞质后的状态及原核形成；最后对猪IVF受精卵受精后8~10 h及ICSI受精卵受精后12~14 h进行EGFP-N1质粒（20 ng/μL）胞质注射，观察胚胎发育效率及转基因效率。结果表明，ICSI受精卵的胚胎发育率（卵裂率89.4%和67.9%、囊胚率36.5%和16.1%）显著优于IVF组（P<0.05），适合用于猪的体外受精卵试验；猪ICSI受精卵双原核在精子注射到卵胞质后12~14 h形成，双原核形成率为54.90%，显著高于其余5个试验组（P<0.05）；ICSI受精卵胞质注射组胚胎卵裂率（86.2%和66.3%）、囊胚率（30.0%和13.6%）及转基因效率（18.5%和0）均显著高于IVF受精卵胞质注射试验组（P<0.05）。本试验结果为采用ICSI受精卵进行胞质注射生产转基因猪的研究奠定了基础。

关键词: 单精子胞浆内注射(ICSI); 胞质注射; 转基因; 猪胚胎

Abstract:

This study was aimed to explore the feasibility of using intracytoplasmic sperm injection (ICSI) method to produce porcine in vitro fertilization (IVF) eggs and application of porcine ICSI fertilized eggs for cytoplasmic injection to produce transgenic embryos.First of all,we compared the efficiency of embryo development between porcine IVF zygotes and ICSI zygotes,and then applied cudbear staining method to observe the pronuclear formation at different time interval and efficiency produced by porcine ICSI zygotes,finally we injected the EGFP-N1(20 ng/μL) plasmid into the zygotes by ICSI and IVF methods to observe the embryonic development efficiency and transgene efficiency.The results showed that the ICSI fertilized eggs had better embryo development rate (cleavage rate 89.4% and 67.9%,blastocyst rate 36.5% and 16.1%) than IVF group(P<0.05);The pronuclear formation time by ICSI method was 12 to 14 h after sperm injection into the cytoplasm,and the rate of prokaryotic nuclei formation was 54.90%,which was significantly higher than the other 5 experimental groups (P<0.05);There was significant difference in the rate of embryonic cleavage rate (86.2% and 66.3%),blastocyst rate (30.0% and 13.6%) and transgene efficiency (18.5% and 0) between ICSI cytoplasmic injection group and IVF test group (P<0.05).This study laid the foundation for the study of producing transgenic pigs by cytoplasmic injection with ICSI fertilized eggs.

Key words: ICSI; cytoplasmic injection; transgene; pig embryos

中图分类号:

S814.8

李红丽, 粟小平, 路粟雨, 刘帅, 刘庆友, 崔奎青, 石德顺. ICSI受精卵胞质注射生产转基因猪胚胎的初步研究[J]. 《中国畜牧兽医》, 2018, 45(6): 1618-1625.

LI Hongli, SU Xiaoping, LU Suyu, LIU Shuai, LIU Qingyou, CUI Kuiqing, SHI Deshun. Study on Transgenic Porcine Embryos by Cytoplasmic Injection into Zygotes Produced Using ICSI Method[J]. , 2018, 45(6): 1618-1625.

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ICSI受精卵胞质注射生产转基因猪胚胎的初步研究

Study on Transgenic Porcine Embryos by Cytoplasmic Injection into Zygotes Produced Using ICSI Method

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