GIMO Transfection in Rodent Malaria Parasites Figure 2. Generation of a marker-free mCherry-expressing parasite using GIMO-transfection. (A) Schematic representation of the introduction of a mCherry-expression cassette into the GIMOPbANKA mother line. Construct pL1628 containing the eef1a-mCherry-39pbdhfr cassette (mCherry; red box) is integrated into the modified P. berghei 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative (Neg) selection with 5-FC selects for parasites (line 1645) that have mCherry reporter introduced into the genome and the hdhfr::yfcu marker removed. Location of primers used for PCR analysis and sizes of PCR products are shown (see Table S2 for primer sequences). (B) Diagnostic PCRs and Southern analysis of PFG-separated chromosomes confirms the correct integration of construct pL1628 in line 1645 parasites shown by the absence of the hdhfr::yfcu marker and the presence of the mCherry gene: 59 integration PCR (59 int; primers 5510/4958), 39 integration PCR (39 int; primers 5515/5511), amplification of hdhfr::yfcu (SM; primers 4698/4699) and the eef1a-mCherry (EF-mC; primers 3173/5514). Primer locations and product sizes are shown in A (primer sequences in Table S2). Hybridization of separated chromosomes of GIMOPbANKA and line 1645 using a hdhfr probe recognizes the hdhfr::yfcu marker in the 230p locus on chromsomse 3 in GIMOPbANKA but is absent in line 1645. Hybridization with 39UTR dhfr probe recognizes both modified the 230p locus on chromosome 3 (both marker and mCherry expression cassettes contain the 39pbdhfr sequence) and the endogenous dhfr/ts gene on chromosome 7 as loading control. (C) Fluorescence microscopy of a live mCherry-expressing trophozoite of line 1645; bright field (BF), DNA staining (Hoechst; Blue) and mCherry expression (red). (D) FACS analysis of mCherry-expressing blood stages of line 1645. The percentage of mCherry-expressing parasites was performed by FACS analysis on cultured blood stage. Mature schizonts (12–16 N) were selected based on their Hoechst fluorescent intensity (gate P2) and mCherry-expressing schizonts were selected in gate P3 (right panel). doi:10.1371/journal.pone.0029289.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29289