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Logo of bmcgeneBioMed Centralsearchsubmit a manuscriptregisterthis articleBMC Genetics
 
BMC Genet. 2001; 2: 21.
Published online Dec 17, 2001. doi:  10.1186/1471-2156-2-21
PMCID: PMC64643
A sensitive and rapid assay for homologous recombination in mosquito cells: impact of vector topology and implications for gene targeting
Paul Egglestoncorresponding author1 and Yuguang Zhao2
1School of Life Sciences, Keele University, Huxley Building, Keele, Staffordshire, ST5 5BG, UK
2Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK
corresponding authorCorresponding author.
Paul Eggleston: p.eggleston/at/keele.ac.uk; Yuguang Zhao: yzhao/at/molbiol.ox.ac.uk
Received October 12, 2001; Accepted December 17, 2001.
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.
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