Shimada Kiyoshi, Ono Tamao, Mizushima Shusei
WCU, Major in Biomodulation, College of Agriculture and Life Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, South Korea.
Faculty of Agriculture, Shinshu University, Kamiina, Nagano 399-4598, Japan.
Gen Comp Endocrinol. 2014 Jan 15;196:100-5. doi: 10.1016/j.ygcen.2013.11.001. Epub 2013 Nov 13.
Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24h at 41°C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12-18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37-50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37°C after the 24h incubation at 41°C under 5% CO2 in air. It survived up to 2days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX-100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24h incubation at 41°C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds.
由于卵母细胞不透明，鸟类的胞浆内精子注射（ICSI）技术受到了阻碍。我们开发了鹌鹑的ICSI辅助受精和基因转移技术。本文综述了我们ICSI实验的最新进展。从输卵管中取出卵母细胞，在体视显微镜下将鹌鹑精子注入卵母细胞。将卵母细胞在41°C、5%二氧化碳的空气中培养24小时。通过显微镜观察评估受精和发育情况。在试验中，受精率为12 - 18%，发育阶段从II期到V期不等。为了提高受精率，将磷脂酶C（PLC）ζ与精子一起注射。受精率提高到了37 - 50%。此外，注射肌醇三磷酸后受精率提高到了85%以上。鹌鹑卵母细胞可以用鸡精子受精，睾丸伸长的精子细胞也可以。为了延长胚胎发育，在41°C、5%二氧化碳的空气中孵育24小时后，将鸡蛋壳用作37°C的替代培养物。此后它存活了2天。最后，尝试在鹌鹑卵中进行基因转移。用Triton X - 100（TX - 100）破坏精子膜，并在卵母细胞中注射PLCζ cRNA和增强型绿色荧光蛋白（EGFP）基因。在41°C、5%二氧化碳的空气中孵育24小时后，在胚胎中评估GFP表达。虽然在对照卵母细胞中未检测到表达，但实验处理诱导了卵母细胞的胚盘发育（44%），86%的胚盘显示荧光发射。此外，PCR分析在50%表达GFP的胚盘中检测到了EGFP片段。我们的ICSI方法可能是生产转基因鸟类的第一步。
