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

Paul Eggleston,Yuguang Zhao

doi:10.1186/1471-2156-2-21

Abstract

BackgroundRecent 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.ResultsWe 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.ConclusionsWe 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.

Highlights

Recent progress in insect transgenesis has been dramatic but existing transposonbased approaches are constrained by position effects and potential instability
Luciferase activities derived from transfection of circular and linear substrates Various combinations of linear and circular substrates were co-transfected into An. gambiae (Ag55) cells and the resulting luciferase activity recorded
We show that linear targeting vectors are better substrates than circular ones and that double-strand breaks stimulate homologous recombination when introduced into, or near, the region of homology

Summary

Introduction

Recent progress in insect transgenesis has been dramatic but existing transposonbased approaches are constrained by position effects and potential instability. There are four transposable element systems (Mos1-mariner, Hermes, Minos and piggyBac) that have been successfully deployed across a range of dipteran, lepidopteran and coleopteran insects [1] This progress has served to focus attention onto potential ap-. We have been investigating the potential for gene targeting in insect genomes through homologous recombination In principle, this could facilitate precise modifications of a given genetic locus provided that the relevant region had previously been cloned [3,4]. This could facilitate precise modifications of a given genetic locus provided that the relevant region had previously been cloned [3,4] This would open the way to gene replacement, knockout or repair, as well as the introduction of specific mutations at the target site. Such precision would greatly enhance the power of investigations into gene function and interaction

Methods

Results

Discussion

Conclusion