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

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.

Fig 1. ΦC31-RMCE in Ae. aegypti.

(a) Schematic diagram of recombination between constructs OX4372 and OX4312 in ΦC31-RMCE, and (b) fourth instar larval expression of fluorescent reported genes in Aedes aegypti OX4372 lines before and after ΦC31 mediated recombination with construct OX4312. OX4372A and OX4372I strains showed only 3xP3-DsRed2 (red eyes). Following recombination in the presence of the ΦC31-integrase, one set of attachment sites resulted in the expression of both 3xP3-DsRed2 (red eyes, shown with white arrows in top right panel) from the docking construct OX4372 and the Hr5ie1-AmCyan (blue bodies) from the donor construct OX4312 (OX4372A[OX4312]-incomplete). Recombination of both sets of ΦC31 attachment sites (attB and attP) resulted in complete RMCE, where only the AmCyan reporter (blue bodies) was observed in OX4372A[OX4312] and OX4372I[OX4312]; the 3xP3-DsRed2 marker for red eyes was excised in these insects (heads are to the left of images; diffuse red staining in larval thorax and abdomen is due to autofluorescence). Scale bars represent 1mm for each image.

Fig 2. iRMCE in Ae. aegypti.

(a) Schematic diagram of iRMCE in Ae. aegypti and (b) corresponding fluorescent phenotypes in fourth instar Ae. aegypti larvae following OX4714 injections. 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. The donor plasmid’s 3xP3-DsRed2 cassette is exchanged for the docking construct’s 3xP3-Amcyan. Larvae are shown under (i) white light, (ii) cyan, and (iii) red excitation light and filters. Images show complete exchange of the 3xP3-AmCyan cassette (OX4476), by integration of 3xP3-DsRed2 (seen in OX4476[OX4714]), and excision of the 3xP3-AmCyan marker (‘excised’ larva). There was reduced expression of the docking cassette’s 3xP3-AmCyan marker following ΦC31-att integration (white arrows: panel (ii), OX4476[OX4714]). Images (i-iii) were taken under the same magnification. Scale bar represents 0.5 mm. White dashed lines outline larvae.

Fig 3. 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.