GIMO Transfection in Rodent Malaria Parasites Figure 6. Compared to the marker-recycling method GIMO-transfection is faster and requires fewer animals to both generate marker-free gene insertion (GI) mutants and to complement gene deletion mutants. (A) Number of weeks (w) and number of mice (m) needed to generate ‘marker-free’ gene insertion mutants expressing transgenes using GIMO-transfection (right) and using the marker-recycling method (left). (B) Number of weeks (w) and number of mice (m) needed for complementation of a gene deletion mutant using GIMO-transfection (right) and using the marker-recycling method (left). doi:10.1371/journal.pone.0029289.g006 construct (see Figure S3 for schematics of the marker-recycling and GIMO methods). In summary, we have developed a novel method that simplifies and speeds up both the generation of marker-free parasites expressing heterologous proteins and for the genetic complemen- tation of gene deletion/mutation mutants. Moreover the applica- tion of this method greatly reduces the numbers of animals required to generate and complement mutants. We have also generated the first marker-free P. yoelii reporter line and established the successful use of negative selection in transfection of P. yoelii parasites. The GIMO-transfection is a simple, fast and efficient approach to generate mutants permissive to subsequent genetic modification. Therefore we recommend that, where possible, transfection of P. berghei and P. yoelii parasites be performed with constructs that contain the postive-negative selectable marker cassette, hdhfr::yfcu. The presence of this marker in mutants permits subsequent GIMO transfection that not only simplifies the creation of additional deletions or modifications but also gene complementation experiments. A recent study has reported high-throughput, genome wide and highly efficient ‘recombineering’ system, for high-throughput, genome wide and highly efficient generation of gene targeting constructs [12]. This PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e29289