显微注射法制备转基因家蝇技术的建立

Preparation of transgenic Musca domestica by microinjection method

摘要:

旨在利用显微注射法对早期家蝇 (Musca domestica L.) 卵注射含有增强型绿色荧光蛋白 (Enhanced green fluorescent protein，EGFP) 基因的转座子载体，实现活体基因稳定表达并对其进行验证，为开展家蝇基因功能的研究奠定基础。文中自制适用于显微注射家蝇卵的硼硅酸盐玻璃微量注射针，摸索出家蝇卵壳的软化处理条件，以Nanoject Ⅲ高精度微量注射器为主体构建适用于家蝇的显微注射技术平台；将含有眼部特异表达的3×P3启动子、EGFP的重组质粒PiggyBac-[3×P3]-EGFP与稳定遗传表达辅助质粒pHA3pig helper显微注射到处理过的家蝇卵中，待羽化观察眼部发光情况，检测EGFP的表达及转录水平。结果表明，将家蝇卵在漂白水中漂洗35 s时卵的正常孵化率为55%，处理35 s的卵壳其硬度适宜注射且注射针头不易破碎；羽化后的家蝇眼部带有绿色荧光的占比约为3%，通过分子检测，家蝇的DNA和RNA中均扩增出EGFP特异片段，大小为750 bp。通过该技术平台，能够便捷、有效地实现报告基因在家蝇中的稳定表达，建立以家蝇为主体的生物反应器，为后续家蝇基因功能的研究提供一定参考价值。

关键词:显微注射，家蝇，受精卵，增强型绿色荧光蛋白，转基因

Abstract:

The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

Key words:microinjection, housefly, fertilized egg, enhanced green fluorescent protein, genetically modified