An efficient and safe strategy for germ cell‐specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms

Abstract

AbstractThe safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1) hybrid eggs produced by cross‐breeding Japanese and Chinese original strains are usually used for the large‐scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad‐specific expression gene promoters (RSHP1p and Nanosp) for the germ cell‐specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4‐Gr and Nsp::GAL4‐Gr, containing the testis‐specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP‐Rg, containing the UAS‐linked FLP gene expression cassette. The FLP recombinase‐mediated sperm‐specific complete excision of FRT‐flanked target DNA in the F1 double‐transgenic silkworms resulting from the hybridization of R1p::GAL4‐Gr and UAS::FLP‐Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4‐Gr and UAS::FLP‐Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad‐specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.