piggyBac-mediated germline transformation of the malaria mosquito Anopheles sinensis (Diptera: Culicidae).

Anopheles sinensis is the most widely distributed species which mainly transmit the Plasmodium vivax malaria in China. Transgenic techniques have been successfully established in many other mosquitoes, but not previously reported in An. sinensis. In this study, the piggyBac transposable element vector pBac[3xP3-EGFP afm] and the piggyBac helper plasmid phspBac were coinjected into preblastoderm eggs of An. sinensis, and the progenies were screened for eye EGFP fluorescence using a fluorescence microscope. A transformation frequency of 21.43% per fertile G0 was obtained. Two transgenic lines, AS2 and AS4, were established and characterized. Maintenance of transformed lines indicates that the transgene has been stable over twelve generations to date. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm] transposon in each transgenic line. The sequence of insertion sites shows the usual canonical pattern of piggyBac integration into TTAA target sites. The eye-specific expression of GFP was continuously observed during larval, pupal and adult stages. Fluorescence was also observed in the neural tube and the anal papillae of larval stages starting from 1 day old larvae in the individuals of both transgenic lines. This work shows that piggyBac can be used for germline transformation in An. sinensis, yielding high transformation frequencies. This efficient method of stable gene transfer in An. sinensis opens the way for promising basic research and new genetics-based methods of control for this malaria vector. This article is protected by copyright. All rights reserved.