Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Biol Reprod. 2010 Dec;83(6):965-9. doi: 10.1095/biolreprod.110.085860. Epub 2010 Aug 18.
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.
本研究旨在建立一种新的通过细胞质内精子注射（ICSI）进行禽类转基因的方法。首先，我们评估了用 PLCZ cRNA 处理的 Triton X-100（TX-100）处理的鹌鹑精子微注射后鹌鹑卵母细胞的受精能力。鹌鹑卵母细胞培养 24 小时后，通过组织学观察检查囊胚发育情况。TX-100 处理会损伤鹌鹑精子膜并干扰注射精子的卵母细胞受精。另一方面，当鹌鹑卵母细胞同时注射 TX-100 处理的精子和 PLCZ cRNA 时，43.5%（23/53）的卵母细胞发育成囊胚。这一发育率与未用 TX-100 处理但用 PLCZ cRNA 处理的精子注射的卵母细胞（14/42.9%）相当。其次，我们评估了在注射 TX-100 处理的精子和 PLCZ cRNA 的鹌鹑卵母细胞中转导增强型绿色荧光蛋白（EGFP）基因的效率。通过胚胎荧光发射的组织学观察评估 EGFP 表达。未用 TX-100 处理但用 PLCZ cRNA 和 EGFP 载体的精子胞质内注射诱导 40%（10/25）的卵母细胞囊胚发育，但这些卵母细胞没有荧光发射。相比之下，TX-100 处理的精子和 PLCZ cRNA 的胞质内注射诱导 43.8%（16/37）的卵母细胞囊胚发育，重要的是，85.7%（6/7）的卵母细胞显示荧光发射。此外，PCR 分析在 50%（3/6）表达 GFP 的囊胚中检测到 GFP 片段。这些结果表明，本文所述的这种额外处理的 ICSI 方法可能是生产转基因鸟类的第一步。
