Highly efficient and inducible DNA excision in transgenic silkworms using the FLP/FRT site-specific recombination system

Abstract

Efficient and inducible recombinase-mediated DNA excision is an optimal technology for automatically deleting unwanted DNA sequences, including selection marker genes. However, this methodology has yet to be established in transgenic silkworms. To achieve efficient and inducible FLP recombinase-mediated DNA excision in transgenic silkworms, one transgenic target strain (TTS) containing an FRT-flanked silkworm cytoplasmic actin 3 gene promoter (A3)-enhanced green fluorescent protein (EGFP) expression cassette, as well as two different types of FLP recombinase expression helper strains were generated. Then, the FLP recombinase was introduced into the TTS silkworms by pre-blastoderm microinjection and sexual hybridization. Successful recombinase-mediated deletion of the A3-EGFP expression cassette was observed in the offspring of the TTS, and the excision efficiencies of the FLP expression vector and FLP mRNA pre-blastoderm microinjection were 2.38 and 13.3 %, respectively. The excision efficiencies resulting from hybridization between the TTS and the helper strain that contained a heat shock protein 70 (Hsp70)-FLP expression cassette ranged from 32.14 to 36.67 % after heat shock treatment, while the excision efficiencies resulting from hybridization between the TTS and the helper strain containing the A3-FLP expression cassette ranged from 97.01 to 100 %. These results demonstrate that the FLP/FRT system can be used to achieve highly efficient and inducible post-integration excision of unwanted DNA sequences in transgenic silkworms in vivo. Our present study will facilitate the development and application of the FLP/FRT system for the functional analysis of unknown genes, and establish the safety of transgenic technologies in the silkworm and other lepidopteran species.

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

“…Further research confirmed that both pre‐blastoderm microinjection of FLP mRNA and continuous expression of FLP recombinase through sexual hybridization can increase the efficiency of the FRT ‐flanked marker gene excision in the transgenic silkworm genome (Long et al. , 2016). We also established a heat shock‐induced site‐specific marker gene excision technology in transgenic silkworms using the FLP/FRT system (Long et al.…”

“…We also established a heat shock‐induced site‐specific marker gene excision technology in transgenic silkworms using the FLP/FRT system (Long et al. , 2016). In 2013, we proved that the phiC31 recombinase‐mediated cassette exchange (phiC31‐RMCE) reaction based on the phiC31/ att system could achieve the precise site‐specific integration/replacement of foreign genes at predetermined chromosome sites in the transgenic silkworm (Long et al.…”

Very Early Corona Treatment-Mediated Artificial Incubation of Silkworm Eggs and Germline Transformation of Diapause Silkworm Strains

“…G0 fertile adults were backcrossed with wild-type adults to produce G1 offspring. The G1 individuals were assessed for the expression of green fluorescent proteins under the stereomicroscope with a GFP filter (Olympus MacroViewMVX10-AUTO, Tokyo, Japan), as described previously (Long et al, 2013;Long et al, 2016).…”

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

“…[38][39][40] The copy number and chromosomal insertion sites of the transgene constructs were determined by Southern blot and inverse PCR, respectively, as described previously with some improvements. 38,39,41 Full experimental procedures are provided in the supplemental information.…”