Novel method of gene transfer in birds: Intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms

Shusei Mizushima, Soichi Takagi, Tamao Ono, Yusuke Atsumi, Akira Tsukada, Noboru Saito, Tomohiro Sasanami, Masaru Okabe, Kiyoshi Shimada

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Original languageEnglish
Pages (from-to)965-969
Number of pages5
JournalBiology of reproduction
Volume83
Issue number6
DOIs
Publication statusPublished - Dec 1 2010
Externally publishedYes

Keywords

  • EGFP
  • ICSI
  • Quail
  • Transgenesis
  • Triton X-100

ASJC Scopus subject areas

  • Reproductive Medicine
  • Cell Biology

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