FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

Abstract

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31

“…The FLP/FRT systems can reportedly mediate site-specific DNA excision and integration in cultured silkworm cells and B. mori [14,17]. Recently, Cre-lox recombinase has also been demonstrated to work in the silkworm [16].…”

“…Although the most commonly used site-specific integrases for genetic manipulation are Cre and FLP, it has been reported that the recombination efficiency is lower when compared with the uC31/att system [5,17,33]. Hence, uC31 integrase is regarded as the best potential tool for transgenesis applications and therapeutic gene transfer [34,35].…”

“…Although SSR technology is able to prevent position effects, caused by random insertion of exogenous DNA, only a few studies have utilized the technique in this species [14][15][16][17]. In this study, we report site-specific gene integration in cultured silkworm cells and B. mori, mediated by uC31 integrase.…”

Advanced technologies for genetically manipulating the silkwormBombyx mori, a model Lepidopteran insect

“…This allows for repeated transgene integration into a site in the genome that will result in predictable transgene expression. These systems have been successfully used in B. mori [19][20][21] although the recombination efficiency has been very low compared with efficiencies seen in other organisms such as Drosophila. Although potentially useful in B. mori, improvements are needed in the performance of these site-specific recombination systems to make them practical options for those working with this species.…”

Genetically engineered mucin mouse models for inflammation and cancer

“…These shortcomings could be overcome by utilizing transgenic (Tg), knockin (KI), and knockout (KO) mouse models, collectively known as genetically engineered mouse (GEM) models. Cre-Lox and Flp-FRT recombination systems are the most commonly used approaches for the development of conditional and inducible KO and KI mouse models [27, 28]. Some of the mucin mouse models have provided crucial information about the importance of mucins including their role under physiological and pathological conditions.…”