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

Abstract

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

Exogenous enzymes upgrade transgenesis and genetic engineering of farm animals

“…Recombination occurs at these pseudo-sites, because of their similarity in nucleotide sequence with the wild-type attP [167]. Pseudo-attP sites have been reported to be present not only in invertebrates [168,169], lower vertebrates [170], but also in mouse [163], human [167,171], cattle [172,173], sheep [174], goat [175] and pig [176] genomes. Accumulating experimental evidence indicates that these pseudo-target sites reside in genomic locations that conform to the definition of ''safe harbors'' [177,178].…”

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

“…However, random insertions of exogenous sequences, resulting from the use of transposase‐mediated transformation systems, may cause unpredictable variations in gene expression and the undesirable mutagenesis of important genes (Labbe et al ., 2010). Furthermore, the recently developed site‐specific recombination systems, for the integration of exogenous genes into the silkworm genome mediated by the phiC31 integrase, provides an alternative that may overcome these problems (Long et al ., 2013; Yin et al ., 2014).…”

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

“…The piggyBac transposon-based germline transformation technology has been extensively applied in functional analyses of B. mori genes and in the development of silkworms as bioreactors (Tamura et al 2000;Tomita et al 2003;Zhao et al 2010). In recent years, an increasing number of genetic manipulation tools have been used successfully in the silkworm, including the Minos transposon (Uchino et al 2007(Uchino et al , 2008, sitespecific recombinase (SSR) systems (Long et al , 2013Duan et al 2013;Yonemura et al 2013;Yin et al 2014), zinc finger nuclease (ZFN) (Takasu et al 2010;Ma et al 2014), transcription-activatorlike effector nuclease (TALEN) Shiomi et al 2015), and clustered regularly-interspersed short palindromic repeats-RNA-guided Cas9 nuclease Zeng et al 2016), to achieve random or site-specific integration of exogenous genes, knockout endogenous genes, and create large genomic deletions.…”

“…The recombination activity of these three recombinases has been demonstrated in B. mori in vivo (Long et al , 2013Duan et al 2013), and these SSR systems have many advantages for transgenic engineering that are lacking in transposonbased systems, including site-specific DNA excision mediated by FLP or Cre recombinase Duan et al 2013) and heritable, site-specific transgene integration mediated by the phiC31 integrase (Long et al 2013;Yonemura et al 2013;Yin et al 2014). Compared with genome-editing nucleases (ZFN,TALEN,and Cas9), the main advantages of SSR systems are that they can be used to achieve precise excision and integration of exogenous DNA fragments at pre-defined chromosomal sites in the silkworm, while genome-editing nucleases are primarily used to knockout endogenous genes (Takasu et al 2010;Wei et al 2014) and excise genomic DNA fragments from B. mori Wang et al 2013); previous studies showed that the efficiencies of ZFN-and TALEN-mediated, site-specific integration of exogenous DNA into the silkworm genome were very low Nakade et al 2014;Wang et al 2014).…”