Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.
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.
Mizushima
Shusei
S
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Takagi
Soichi
S
Ono
Tamao
T
Atsumi
Yusuke
Y
Tsukada
Akira
A
Saito
Noboru
N
Sasanami
Tomohiro
T
Okabe
Masaru
M
Shimada
Kiyoshi
K
D000818:Animals; D030801:Animals, Genetically Modified; D001756:Blastoderm / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects; D003370:Coturnix / Q000196:embryology / Q000235:genetics*; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro / Q000187:drug effects / Q000662:veterinary; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D017830:Octoxynol / Q000494:pharmacology; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics; D020554:Sperm Injections, Intracytoplasmic / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects; D013501:Surface-Active Agents / Q000494:pharmacology
D018075:RNA, Complementary; D013501:Surface-Active Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017830:Octoxynol; D054801:Phosphoinositide Phospholipase C