Germ line transformation of the olive fly Bactrocera oleae using a versatile transgenesis marker

Abstract

The olive fruit fly (olive fly) Bactrocera oleae (Dacus), recently introduced in North America, is the most destructive pest of olives worldwide. The lack of an efficient gene transfer technology for olive fly has hampered molecular analysis, as well as development of genetic techniques for its control. We have developed a Minos-based transposon vector carrying a self-activating cassette which overexpresses the enhanced green fluorescent protein (EGFP). Efficient transposase-mediated integration of one to multiple copies of this vector was achieved in the germ line of B. oleae by coinjecting the vector along with in vitro synthesized Minos transposase mRNA into preblastoderm embryos. The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis of very diverse species.

Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration.

“…This strategy requires a co-delivery of pDNA encoding the therapeutic gene and the transposase mRNA. The Minos-based transposon system has been shown to be active in various organisms, with, for example, efficient transposase insertion of one or multiple copies into the germ line of Bactrocera oleae [116]. This system is also active in mammalian tissues [118].…”

“…Transposon-based systems including Piggyback, Sleeping Beauty and Minos transposons are promising tools for the integration of a gene of interest into the genome [113][114][115][116]. Transposon systems direct the precise transfer of specific parts of a donor pDNA into a mammalian chromosome.…”

Isolation, annotation and applications of expressed sequence tags from the olive fly, Bactrocera oleae

“…In the past decade, molecular and genetic studies have focused on genetic analysis of natural populations (Augustinos et al 2005;Nardi et al 2005;Zygouridis et al 2009), cytogenetics (for a review see Mavragani-Tsipidou 2002), isolation and characterization of a few genes that control important processes such as female germline diVerentiation and morphogenesis of epidermal cells (Khila et al 2003), enzyme catalytic mechanisms (Benos et al 2000) and sexdetermining cascades (Lagos et al 2005(Lagos et al , 2007 as well as the identiWcation and mapping of several microsatellite loci (Augustinos et al 2002(Augustinos et al , 2005(Augustinos et al , 2008. Most notably, B. oleae has been successfully transformed recently (Koukidou et al 2006), an achievement that gives new perspectives towards the eYcient use of the SIT. Nonetheless, there is still an absence of genetic markers and promoter information that will render transformation more useful and eVective.…”

Safe and fit genetically modified insects for pest control: from lab to field applications

“…The development and implementation of the SIT against this species has been so rapid and effective that it has been validated and practiced on industrial and area-wide scale (Hendrichs 2000;Klassen and Curtis 2005). In addition, the medfly was the first non-drosophilid insect to be transformed (Loukeris et al 1995) and this achievement opened the way to the genetic transformation of many other pest insects that are targets of SIT programmes (Handler and McCombs 2000;Handler and Harrell 2001;Handler and O'Brochta 2005;Koukidou et al 2006;Condon et al 2007a). Different medfly strains are currently available that should increase the efficacy and cost effectiveness of the SIT both at the mass-rearing, releasing and monitoring stages, since they provide (1) genetic marking for the identification of transformed insects (Zwiebel et al 1995;Handler et al 1998;Michel et al 2001); (2) male-specific fluorescent sorting (Scolari et al 2008b); (3) sexing for male-only strains Condon et al 2007b); (4) reproductive sterility through embryonic lethality (Gong et al 2005;Schetelig et al 2009a).…”