A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting

Abstract

Background: Recent progress in insect transgenesis has been dramatic but existing transposon-based approaches are constrained by position effects and potential instability. Gene targeting would bring a number of benefits, however progress requires a better understanding of the mechanisms involved. Much can be learned in vitro since extrachromosomal recombination occurs at high frequency, facilitating the study of multiple events and the impact of structural changes among the recombining molecules. We have investigated homologous recombination in mosquito cells through restoration of luciferase activity from deleted substrates. The implications of this work for the construction of insect gene targeting vectors are discussed.

Results: We show that linear targeting vectors are significantly more efficient than circular ones and that recombination is stimulated by introducing double-strand breaks into, or near, the region of homology. Single-strand annealing represents a very efficient pathway but may not be feasible for targeting unbroken chromosomes. Using circular plasmids to mimic chromosomal targets, one-sided invasion appears to be the predominant pathway for homologous recombination. Non-homologous end joining reactions also occur and may be utilised in gene targeting if double-strand breaks are first introduced into the target site.

Conclusions: We describe a rapid, sensitive assay for extrachromosomal homologous recombination in mosquito cells. Variations in substrate topology suggest that single-strand annealing and one-sided invasion represent the predominant pathways, although non-homologous end joining reactions also occur. One-sided invasion of circular chromosomal mimics by linear vectors might therefore be used in vitro to investigate the design and efficiency of gene targeting strategies.

Figure 1

Design and construction of the recombination substrates. p[ACT-LUC] carries an intact transcription unit comprising the firefly (Photinus pyralis) luciferase coding sequence (red) driven from the D. melanogaster actin5C promoter (blue) with transcription terminated by the SV40 small t intron/polyA signal (yellow). The luciferase coding sequence was released from pGEM-luc (Promega) by digestion with BamHI and XhoI and ligated into the same sites located between the actin5C promoter and SV40 termination sequence in p[ACT-SV] (unpublished data). All relevant restriction enzyme sites are indicated. The left-hand deletion substrate (DL) was generated by XbaI and BstEII digestion to remove a 561 bp fragment at the 5' end of the luciferase coding sequence. The right-hand deletion substrate (DR) was generated by EcoRV and XhoI digestion to remove a 371 bp fragment at the 3' end of the luciferase coding sequence. The 728 bp region of homology shared by DL and DR is indicated and homologous recombination in this interval has the potential to reconstitute a functional luciferase gene.

Figure 2

Functional and molecular analysis of extrachromosomal homologous recombination. The upper panel is a schematic of the left and right-hand deletion substrates (DL and DR) showing the actin5C promoter (blue), luciferase coding sequence (red) and SV40 termination sequence (yellow). The 5' and 3' luciferase deletions are shown as dotted lines and the pUC18 plasmid backbone as a continuous line. The intact luciferase coding sequence used as a probe for Southern analysis is shown in green. All relevant restriction sites are indicated (B, BamHI; S, SacI; H, HindIII and Sc, ScaI). The central panel shows the various combinations of plasmids transfected into mosquito cells together with a Southern analysis of the reaction products in An. gambiae (Ag55) cells. Transfections involved either circular (C) or linear (L) forms of the parent plasmid (p[ACT-LUC]) and deletion substrates (DL; DR), as shown, with superscripts indicating linearization by the relevant restriction enzyme (L^B, BamHI; L^S, SacI; L^H, HindIII; L^Sc, ScaI). For Southern analysis, total cellular DNA was isolated from An. gambiae (Ag55) cells 48 hours post-transfection, digested with both BamHI and BglII, resolved on 1.5% agarose and blotted onto nitrocellulose. The membrane was probed with a ³²P labelled luciferase fragment, washed at high stringency (1 × SSC; 0.1 % SDS; 65°C) and exposed overnight at -70°C to X-ray film against an intensifying screen. The size of relevant signals was determined by comparison to standard markers (MBI Kilobase Ladder) and is shown in kilobase pairs (Kb). The lower panel shows the mean luciferase activities (log₁₀ counts per minute) recovered from the various transfections into An. gambiae (Ag55) cells. Values were plotted following subtraction of the assay background and the error bars represent standard deviations. A logarithmic scale was employed so that all values could be represented on the same figure.

Figure 3

Comparative analysis of recovered luciferase activities across cell lines. Mean luciferase activities (log₁₀ cpm) recovered from the various transfections in cell lines derived from Ae. aegypti (Mos20; blue), An. gambiae (Ag55; magenta), Culex pipiens pallens (Cpp512, yellow), An. stephensi (As43, green) and An. sinensis ovary (Anso, red). Standard deviations are not shown for reasons of clarity but were comparable to those shown in Fig. 2 for An. gambiae (Ag55) cells. Transfections involved either circular (C) or linear (L) forms of the parent plasmid (p[ACT-LUC]) and deletion substrates (DL; DR), as shown, with superscripts indicating linearization by relevant restriction enzyme (L^B, BamHI; L^S, SacI; L^H, HindIII; L^Sc, ScaI). A logarithmic scale was employed so that all values could be represented on the same figure.