A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites

Abstract

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel 'gene insertion/marker out' (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research.

Figure 1. Generation and genotype analyses of P. berghei and P. yoelii GIMO mother lines.

(A) Schematic representation of the constructs used to introduce the positive-negative selectable maker cassette in the P. berghei (PbANKA) or P. yoelii (Py17XNL) 230p locus. DNA constructs pL1603 (targeting P. berghei 230p, PBANKA_030600) and pL1805 (targeting P. yoelii 230p, PY03857) containing a fusion of the positive drug selectable marker hdhfr (human dihydrofolate reductase) and negative marker yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase) under the control of the eef1α promoter target the 230p locus at the target regions (hatched boxes) by double cross-over homologous recombination. Location of primers used for PCR analysis and sizes of PCR products are shown (see Table S2 for all primer sequences). (B) Diagnostic PCR and Southern analysis of PFG-separated chromosomes confirming correct integration of the construct in the P. berghei mother line GIMO_PbANKA: 5′ integration PCR (5′ int; primers 5510/3189), 3′ integration PCR (3′ int; primers 4239/5511), amplification of hdhfr::yfcu marker (SM; primers 4698/4699) and the original P. berghei 230p (230p; primers 1637/5600). Primer location (black arrows) and product sizes are shown in A. For Southern analysis, PFG-separated chromosome were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei 230p locus on chromosome 3 and the endogenous locus of dhfr/ts on chromosome 7. (C) Diagnostic PCR and Southern analysis of PFG-separated chromosomes confirming correct integration of the construct in the P. yoelii mother line GIMO_Py17X: 5′ integration PCR (primers 6527/4770), 3′ integration PCR (primers 4771/6528), amplification of hdhfr::yfcu marker (primers 4698/4699) and the P. yoelii 230p original locus (primers 6529/6530). Primer location (grey arrows) and product sizes are shown in A. For Southern analysis, chromosomal hybridization using a 3′UTR pbdhfr probe recognizes the construct integrated into P. yoelii 230p locus on chromosome 3 and the endogenous locus of dhfr/ts on chromosome 7.

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 GIMO_PbANKA mother line. Construct pL1628 containing the eef1α-mCherry-3′pbdhfr 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: 5′ integration PCR (5′ int; primers 5510/4958), 3′ integration PCR (3′ int; primers 5515/5511), amplification of hdhfr::yfcu (SM; primers 4698/4699) and the eef1α-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 GIMO_PbANKA and line 1645 using a hdhfr probe recognizes the hdhfr::yfcu marker in the 230p locus on chromsomse 3 in GIMO_PbANKA but is absent in line 1645. Hybridization with 3′UTR dhfr probe recognizes both modified the 230p locus on chromosome 3 (both marker and mCherry expression cassettes contain the 3′pbdhfr 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).

Figure 3. The efficiency of GIMO-transfection to select marker-free parasites that express mCherry.

(A) Percentage of mCherry-positive parasites in GIMO-transfection of GIMO_PbANKA (shown in Figure 2) after negative selection. The percentage of mCherry-positive parasites in six independent transfections (1794–1799) was determined by FACS analysis (see Figure 2D) and quantitative PCR (qPCR). By qPCR the ratio of mCherry and hdhfr::yfcu marker positive parasites was determined relative to the presence of a control gene hsp70, using the 2^−ΔΔCT method (primers used in qPCR are described in Table S2). (B) Efficiency of selection of hdhfr::yfcu marker-free determined by Southern analysis of PFG-separated chromosomes. Hybridization performed using a mixture of two probes, one specific for pb25 (chromosome 5) and one for hdhfr (chromosome 3) showing the efficiency of selecting hdhfr::yfcu marker-free parasites in the different experiments.

Figure 4. Generation of a P. yoelii reporter line, PyGFP-luc_con that is marker-free and expresses a fusion protein of GFP and luciferase.

(A) Schematic representation of the introduction of a gfp-luciferase-expression cassette into the GIMO_Py17X mother line. Construct pL1847 containing the eef1α-gfp::luciferase-3′pbdhfr cassette is integrated into the modified P. yoelii 230p locus containing the hdhfr::yfcu selectable marker cassette (black box) by double cross-over homologous recombination at the target regions (hatched boxes). Negative selection with 5-FC results in selection of parasites that have the gfp-luciferase 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) Fluorescence microscopy of a live schizont of PyGFP-luc_con; bright field (BF), DNA staining (Hoechst; Blue) and GFP expression (green). (C) PFG-separated chromosomal Southern analysis of two independent GIMO transfection parasite lines (exp. 1970 and 1971). Hybridization performed with a mixture of two probes, one specific for pb25 (chromosome 5) and the other for hdhfr (chromosome 3), demonstrating the efficiency of selection of hdhfr::yfcu ‘marker-free’ parasites in the different experiments. (D) Analysis of luciferase-expression of blood stages of 3 clones of PyGFP-luc_con (exp. 1971). Luciferase-activity was measured by real time in vivo imaging of live mice with a parasitemia of 1–3%. (E) Diagnostic PCR analysis confirming correct integration of the gfp-luciferase gene in PyGFP-luc_con clones (exp. 1971): amplification of hdhfr::yfcu marker (SM, primers 4698/4699), 5′ integration PCR (5′ int, primers 6527/6812), 3′ integration PCR (3′ int, primers 6813/6528) and gfp-luc (primers 6814/6815). Primer location, product sizes are shown in A and primer sequences in Table S2.

Figure 5. Gene complementation using GIMO-transfection.

(A) Schematic representation of the re-introduction of the glutathione reductase (gr) gene into the gr gene deletion mutant (Δgr, 1513cl1); 1513cl1 expresses the hdhfr::yfcu selectable marker (black box). Transfection with a 2.8 kb PCR-fragment amplified from wild type genomic DNA (primers 4530/3681) containing the gr gene, as well as the 5′- and 3′-targeting sequences, was used to re-introduce gr gene into the Δgr mutant. Negative selection with 5-FC selects for parasites that have the gr gene re-introduced into the genome replacing the hdhfr::yfcu marker (line 1761; Δgr(+gr). Location of primers used for PCR analysis, sizes of PCR products, restriction enzyme sites and sizes of the expected fragments in Southern analysis are indicated (see Tables S1 and S2 for primer sequences). (B) Diagnostic PCR analysis and Southern analysis of restricted genomic DNA confirm correct integration of the PCR fragment and complementation in Δgr(+gr) parasites: amplification of hdhfr::yfcu marker (SM; primers 4698/4699) and gr (ORF; primers 3742/3743). Primer location, product sizes are shown in A and primer sequences in Table S2. Southern blot was hybridized with 3′UTR gr probe (i.e. 3′ targeting region). The localization of the restriction enzymes used and the expected size of the fragments are shown in A: wt (wild type); Δgr (gr deletion mutants); Δgr(+gr) (complemented Δgr); mp (blood stages after mosquito passage). (C) Oocyst development of Δgr and Δgr (+gr) parasites. Only small, aberrant oocysts with no signs of sporozoite formation are present in Δgr infected mosquitoes at days 10–21 after feeding. In Δgr (+gr) infected mosquitoes sporozoite-containing oocysts with wild-type morphology are visible at day 12. (E) Salivary gland sporozoites of Δgr(+gr) examined by immuno-fluorescence microscopy: bright field (BF) and anti-CS antibody staining (CS, green).

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).