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Site-specific cassette exchange systems in the Aedes aegypti mosquito and the Plutella xylostella moth.

Haghighat-Khah RE, Scaife S, Martins S, St John O, Matzen KJ, Morrison N, Alphey L - PLoS ONE (2015)

Bottom Line: Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms.We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes.A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom; Oxitec Limited, Oxford, United Kingdom.

ABSTRACT
Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests.

No MeSH data available.


Related in: MedlinePlus

iRMCE in P. xylostella.(a) Schematic diagram of iRMCE in P. xylostella and (b) corresponding fluorescent phenotypes in P. xylostella pupae. Black arrows indicate engineered FRT and loxP sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate piggyBac ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.
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pone.0121097.g003: iRMCE in P. xylostella.(a) Schematic diagram of iRMCE in P. xylostella and (b) corresponding fluorescent phenotypes in P. xylostella pupae. Black arrows indicate engineered FRT and loxP sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate piggyBac ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.

Mentions: Plutella xylostella. A total of 1082 embryos from the docking strain OX4540A were injected with OX4580 and ΦC31-integrase mRNA; of these, 332 individuals survived to pupation. Crosses to wild-type were set up at the pupal stage. Their progeny were screened for ZsGreen expression (carried by OX4580). Out of 91 crosses, one generated individuals expressing ZsGreen. These were used to establish line OX4580[4540A] (Fig 3). The minimum calculated integration efficiency was 0.30%. Similar integration efficiencies (0.16% and 0.39%) for ΦC31 integration in P. xylostella were obtained previously in our lab (data not shown).


Site-specific cassette exchange systems in the Aedes aegypti mosquito and the Plutella xylostella moth.

Haghighat-Khah RE, Scaife S, Martins S, St John O, Matzen KJ, Morrison N, Alphey L - PLoS ONE (2015)

iRMCE in P. xylostella.(a) Schematic diagram of iRMCE in P. xylostella and (b) corresponding fluorescent phenotypes in P. xylostella pupae. Black arrows indicate engineered FRT and loxP sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate piggyBac ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382291&req=5

pone.0121097.g003: iRMCE in P. xylostella.(a) Schematic diagram of iRMCE in P. xylostella and (b) corresponding fluorescent phenotypes in P. xylostella pupae. Black arrows indicate engineered FRT and loxP sites present in donor and acceptor constructs; AmpR represents the plasmid backbone sequences, which includes the ampicillin-resistance gene; grey arrows indicate piggyBac ends. Pupae are shown under (i) white light, (ii) green excitation light and filters, and (iii) red excitation light and filters. Images (i–iii) were taken under the same magnification. Scale bar repersents 1 mm.
Mentions: Plutella xylostella. A total of 1082 embryos from the docking strain OX4540A were injected with OX4580 and ΦC31-integrase mRNA; of these, 332 individuals survived to pupation. Crosses to wild-type were set up at the pupal stage. Their progeny were screened for ZsGreen expression (carried by OX4580). Out of 91 crosses, one generated individuals expressing ZsGreen. These were used to establish line OX4580[4540A] (Fig 3). The minimum calculated integration efficiency was 0.30%. Similar integration efficiencies (0.16% and 0.39%) for ΦC31 integration in P. xylostella were obtained previously in our lab (data not shown).

Bottom Line: Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms.We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes.A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Oxford, Oxford, United Kingdom; Oxitec Limited, Oxford, United Kingdom.

ABSTRACT
Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests.

No MeSH data available.


Related in: MedlinePlus