High efficiency site-specific genetic engineering of the mosquito genome

Abstract

Current techniques for the genetic engineering of insect genomes utilize transposable genetic elements, which are inefficient, have limited carrying capacity and give rise to position effects and insertional mutagenesis. As an alternative, we investigated two site-specific integration mechanisms in the yellow fever mosquito, Aedes aegypti. One was a modified CRE/lox system from phage P1 and the other a viral integrase system from Streptomyces phage phi C31. The modified CRE/lox system consistently failed to produce stable germline transformants but the phi C31 system was highly successful, increasing integration efficiency by up to 7.9-fold. The ability to efficiently target transgenes to specific chromosomal locations and the potential to integrate very large transgenes has broad applicability to research on many medically and economically important species.

Figure 1

Genomic analysis of attP target sites in five transgenic strains (17, 13 W, 13, 12, 11 and 2) of Aedes aegypti generated by piggyBac-mediated germline transformation. The suffix ‘W’ in strain 13 W indicates a white-eyed phenotypic background. Strain 13 W carries the same attP target site as strain 13 and was generated by crossing strain 13 (wild-type eye colour) to the kh^w white-eyed mutant strain, followed by selection of individuals homozygous for the white mutation. (a) Southern blot of genomic DNA (10 μg) digested with EcoRV and probed at high stringency with the fragment indicated in Fig. 2(a). Three of the strains (2, 12, and 13/13 W) have a single attP site, strain 11 has two attP sites and strain 17 has four attP sites. The wild-type host (WT) is devoid of attP sites. (b) Inverse PCR analysis of attP locations showing mosquito genomic sequence at the 5′-and 3′ flanks of the piggyBac integrations. All insertion sites are unique and all have the characteristic TTAA sequence duplication. The four integration sites in strain 17 could not be resolved by inverse PCR. The 3′ flanking sequence obtained for strain 13 by inverse PCR on DpnII restricted DNA was foreshortened by the presence of a DpnII restriction site seven nucleotides downstream of the TTAA motif.

Figure 2

phi C31-mediated site-specific transgene integration in Aedes aegypti. (a) chromosomal organization of the targeting strains (not to scale) showing an unoccupied attP target site inserted by piggyBac and marked by ECFP. pB-L and pB-R are the left and right piggyBac terminal inverted repeats and the black double arrow is the probe used in Fig. 1(a) to identify attP sites. Short arrows show the location of PCR primers that amplify a 391 bp fragment specific for attP. (b) chromosomal organization at the occupied target site (not to scale) following site-specific integration of pBattB[3 × P3-DsRed2nls-SV40]lox66. The black double arrow is the probe used to detect site-specific integration in Fig. 2(c). Short arrows show the location of PCR primers that amplify 224 bp and 301 bp fragments specific to attR and attL, respectively. Relevant restriction sites are XbaI (X), EcoRV (E), PstI (P), NotI (N) and SalI (S). (c) Southern blot analysis of three targeting strains (17, 12 W and 11) both before and after (*) site-specific transgene integration. Genomic DNA (10 μg) was digested with XbaI and probed at high stringency with the fragment identified in Fig. 2(b). In all strains, a 1527 bp band that defines the empty attP target site increases to 2698 bp following site-specific integration of pBattB[3 × P3-DsRed2nls-sv40]lox66. (d) PCR amplification with primers specific for attP identifies a 391 bp product that defines the unoccupied target site. Following site-specific integration, primers specific for attL and attR identify characteristic products of 301 bp and 224 bp, respectively.

Figure 3

Fluorescence profiles of transgenic Aedes aegypti showing phi C31-mediated site-specific transgene integration. Site-specific integration in strain 12 W (white-eyed background) is evident from colocalization of the ECFP (a) and DsRed2 (b) fluorophores in the eyes, optic nerves and antennae. Strain 11 (wild-type eye background) (c,d,e,f) exhibits fluorescence in the anal papillae as well as in the eyes and optic nerves and this emphasizes the colocalization of fluorophores following site-specific integration. Note that the ECFP (cyan) fluorescence profile (c, e) is mirrored exactly by that of DsRed2 (d, f). Note also that DsRed2 expression is restricted to the cell nuclei by a nuclear localization signal.