High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

Jasinskiene, Nijole; Coates, Craig J.; Ashikyan, Aurora; James, Anthony A.

doi:10.1093/nar/gng148

Cited by 31 publications

(21 citation statements)

References 26 publications

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“…In particular, the functionality of piggyBac-based transformation systems and fluorescent transformation marker genes has been demonstrated in a broad spectrum of insect species (23,37). Although FLP activity in embryos of the silkworm Bombyx mori and the mosquito Aedes aegypti could be demonstrated (38,39), FLP-mediated excision of a FRT-flanked marker gene from the mosquito chromosome could not be detected (40). Thus, the activity of FLP recombinase, as well as the behavior of Drosophila linotte or linotte-homologous sequences in insect species of interest, remains to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Site-specific genomic targeting inDrosophila

Horn

Handler

2005

Proc. Natl. Acad. Sci. U.S.A.

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Transcriptional regulation of transgenes depends upon genomic localization in higher eukaryotes. For the applied use of transgenic organisms as producers of pharmaceutically relevant proteins or as pest population control agents, a method to make transgene expression predictable is highly desirable. A targeting method that allows precise cassette replacement comprising solely genes of interest (without extraneous donor vector sequences) would be highly advantageous for insects and other multicellular organisms. In this report, we describe a method for transgene targeting to predefined chromosomal sites in Drosophila by using a transposon vector that, once integrated in the germ line, acts as an acceptor site for donor vectors. To make recombinational insertions irreversible, a FLP recombinase-mediated cassette exchange strategy was used, and to enhance donor-target pairing, a homing sequence from the linotte locus was used. Site-specific recombinants were screened by interconvertible eye fluorescence marker phenotypes yielding, on average, targeted insertions at a frequency of 23%. The cassette exchange system provides for repetitive integrations into the same locus, allowing comparative analysis of true transgenic alleles. Furthermore, this method was used to stabilize a targeted transgene by the postintegration excision of putatively mobile transposon sequences. The genomic targeting and stabilization strategy described for Drosophila should be applicable to other insects, specifically for the goals of optimizing heterologous protein expression and enhancing ecological safety of transgenic strains intended for release in biocontrol programs.homing sequence ͉ insect transformation ͉ recombinase-mediated cassette exchange ͉ transgene stabilization S ite-specific recombinases such as FLP, Cre, and ⌽C31 have emerged as powerful tools to manipulate genomes of eukaryotic model organisms (1). In mice, site-specific recombinase technology is being applied to modify chromosomal DNA in a spatially and temporally controlled manner, thereby bypassing embryonic lethality associated with many germ-line null alleles (2). In Drosophila, the FLP͞FRT (FRT, FLP recombinase target site) (3) has been extensively used to generate genetic mosaics in soma and germ-line (4), and chromosome rearrangements (5). Recently, systematic collections of molecularly defined deletions spanning Ͼ50% of the Drosophila melanogaster genome have been engineered by using FLP͞FRT (6, 7).Site-specific recombinase proteins catalyze the cutting and rejoining of two different DNA segments at specific target sequences. Depending on the relative orientation of the recombinase target sites, the outcome of a recombination reaction is excision and insertion of a circular DNA molecule, respectively, or inversion of intervening DNA. Excision of genomic DNA located between two equally oriented target sites is effectively irreversible due to the creation and, in dividing cells, loss of an episomal reaction product. In contrast, integration of a plasmid at a genomic...

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Section: Discussionmentioning

confidence: 99%

Site-specific genomic targeting inDrosophila

Horn

Handler

2005

Proc. Natl. Acad. Sci. U.S.A.

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“…The characterization of mosquito promoter sequences could be greatly aided by utilization of the Cre-LoxP system, which was shown recently to be active in Ae. aegypti (Jasinskiene et al, 2003). This system would allow precise functional comparison of alternative promoters if it is used to target integration of transgenes into the same chromosomal location: vagaries in expression caused by position effects of the transgene insertion site would be eliminated, and promoter activity would be easily compared between transgenic lines.…”

Section: Direct and Heritable Reverse Geneticsmentioning

confidence: 99%

Innate immunity in the malaria vectorAnopheles gambiae:comparative and functional genomics

Osta

Christophides

Vlachou

et al. 2004

Journal of Experimental Biology

118

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“…Cre- loxP excision was previously shown to be highly efficient for removal of a marker from transgenic Ae. aegypti [47]. Excision could therefore be accomplished by the inclusion of loxP sites either side of the phase 1 integrase and marker cassettes.…”

Section: Discussionmentioning

confidence: 99%

Next-Generation Site-Directed Transgenesis in the Malaria Vector Mosquito Anopheles gambiae: Self-Docking Strains Expressing Germline-Specific phiC31 Integrase

et al. 2013

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Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.

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High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti

Cited by 31 publications

References 26 publications

Site-specific genomic targeting inDrosophila

Site-specific genomic targeting inDrosophila

Innate immunity in the malaria vectorAnopheles gambiae:comparative and functional genomics

Next-Generation Site-Directed Transgenesis in the Malaria Vector Mosquito Anopheles gambiae: Self-Docking Strains Expressing Germline-Specific phiC31 Integrase

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