Preparation of transgenic Musca domestica by microinjection method.

Lanchen Wang; Yang Yang; Xiaoli Shang; Bing Wang; Lin Yuan; Guiming Zhu

Return

Preparation of transgenic Musca domestica by microinjection method.

Author: Lanchen WANG ¹ ; Yang YANG ¹ ; Xiaoli SHANG ² ; Bing WANG ¹ ; Lin YUAN ² ; Guiming ZHU ¹
Author Information

1. Key Laboratory of Biotechnology and Medical Engineering, School of Biology and Engineering, Guizhou Medical University, Guiyang 550025, Guizhou, China.
2. Biotechnology Engineering Research Center, School of Biology and Engineering, Guizhou Medical University, Guiyang 550025, Guizhou, China.
Publication Type:Journal Article
Keywords: enhanced green fluorescent protein; fertilized egg; genetically modified; housefly; microinjection
MeSH: Animals; Animals, Genetically Modified; Gene Expression; Genes, Reporter; Green Fluorescent Proteins/genetics*; Houseflies/genetics*; Microinjections
From: Chinese Journal of Biotechnology 2021;37(2):655-662
CountryChina
Language:Chinese
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.