Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

Abstract

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

Ras1CA overexpression in the posterior silk gland improves silk yield

“…Approximately 10 years ago, a stable germ-line transformation method was established for the silkworm using the transposable element piggyBac as the transgenic vector [14]. Based on this method, a binary GAL4/UAS system for targeted gene expression was developed [11] and was further applied to analyze gene function of juvenile hormone esterase [12].…”

“…T h e p B a c { F i l -G A L 4 -3 X P 3 -D s R e d } ( F i l -G A L 4 ) a n d pBac{UAS-Ras1 v12 -3XP3-EGFP} (UAS-Ras1 CA ) plasmid constructs (Supplementary information, Figure 3A) were generated as described below with the transposable element piggyBac as the transgenic vector [14]. The 740-bp promoter region of the fibroin light chain (Fil) gene [11,28] was amplified using forward and reverse primers containing AscI and BamHI sites, respectively.…”

“…The BamHI-SacII fragment, which contained the GAL4 gene and Dmhsp70 terminator, was amplified from phsGAL4 [29]. The above two PCR fragments were conjugated, digested with AscI and SacII, and inserted into pBac{3XP3-DsRed} [14] to generate Fil-GAL4. The cDNA of Bombyx Ras1 oncogene [9] was amplified using forward primer containing EcoRI and reverse primer containing NotI.…”

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…Despite strong interest in this group, however, there has been a frustrating lack of progress in developing routine approaches for manipulative genetic work. While the last two decades have seen examples of transgenesis and targeted knockouts using methods like transposon insertion (Tamura et al 2000), zinc-finger nucleases (Takasu et al 2010;Merlin et al 2013), and TALENs (Takasu et al 2013;Markert et al 2016), especially in the silk moth Bombyx mori, these approaches have resisted widespread application due to their laborious nature. We see two other main reasons manipulative genetics has failed to become routine in Lepidoptera.…”

Relations between animal transgenesis and reproduction

“…Transposon P is extensively used to generate transgenic Drosophilae. The transposons Sleeping Beauty and piggy Bac are used for a number of species [2] and for the silk worm [3] respectively. Other transposons are used for various species of insects.…”