Studying gene function in the human malaria vector Anopheles gambiae is the key to understanding its biology and vector-parasite interactions. Existing tools to study gene function do not include gene targeting, which would allow insights into gene function by permitting a range of specific modifications to any gene of choice.
Based on developments in Drosophila (RONG and GOLIC 2000) this PhD project proposes to establish a method for gene targeting in Anopheles by homologous recombination, using a linear targeting construct generated in vivo. To this end, a transgenic strain was generated that expresses FLP recombinase and I-SceI endonuclease under the control of the germline-specific vasa promoter. Together they were to excise and linearise a targeting molecule from a transgenic “donor” locus. Homology in the targeting construct would enable integration, via recombination in the germline, at a gene of interest, thereby permitting its targeted modification.
Expression of vasa-driven I-SceI resulted in high cleavage activity in the germline. A systematic analysis using a variety of transgenic target loci revealed that homologous repair, rather than non-homologous end joining, was the predominant mechanism employed to repair the double stranded breaks generated by I-SceI. These findings offer encouraging prospects for population genetic engineering using homing endonuclease genes (BURT 2003). FLP activity was shown in Anopheles cell culture, yet in vivo excision via FLP at two independent loci in the germline was not observed, precluding the obtainment of a knock-out. After eliminating many possible sources of error, likely causes include antagonistic interference between I-SceI and FLP or unfavourable reaction kinetics of FLP.