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

Biol Reprod. 2010 Dec;83(6):965-9. doi: 10.1095/biolreprod.110.085860. Epub 2010 Aug 18.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Blastoderm / cytology
  • Blastoderm / metabolism*
  • Cell Membrane / drug effects
  • Coturnix / embryology
  • Coturnix / genetics*
  • Embryo Culture Techniques / veterinary
  • Embryonic Development / drug effects
  • Female
  • Fertilization in Vitro / drug effects
  • Fertilization in Vitro / veterinary
  • Gene Expression*
  • Gene Transfer Techniques / veterinary*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Male
  • Octoxynol / pharmacology
  • Phosphoinositide Phospholipase C / genetics
  • Phosphoinositide Phospholipase C / metabolism
  • RNA, Complementary / genetics
  • Sperm Injections, Intracytoplasmic / veterinary*
  • Spermatozoa / drug effects
  • Surface-Active Agents / pharmacology

Substances

  • RNA, Complementary
  • Surface-Active Agents
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Octoxynol
  • Phosphoinositide Phospholipase C