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    Thermo Fisher pcr amplification
    Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range <t>PCR</t> analysis of genomic <t>DNA</t> from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009
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    Thermo Fisher pcr fragments
    Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range <t>PCR</t> analysis of genomic <t>DNA</t> from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009
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    Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009

    Journal: eLife

    Article Title: A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites

    doi: 10.7554/eLife.03582

    Figure Lengend Snippet: Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009

    Article Snippet: All DNA fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen, Leek, The Netherlands) sub-cloning.

    Techniques: Generated, Polymerase Chain Reaction, Marker, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Purification, Positive Control

    Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006

    Journal: eLife

    Article Title: A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites

    doi: 10.7554/eLife.03582

    Figure Lengend Snippet: Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006

    Article Snippet: All DNA fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen, Leek, The Netherlands) sub-cloning.

    Techniques: Mutagenesis, Construct, Generated, Marker, Sequencing, Diagnostic Assay, Polymerase Chain Reaction, Luciferase, In Vivo Imaging, Expressing, Mouse Assay, Infection, Injection

    Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009

    Journal: eLife

    Article Title: A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites

    doi: 10.7554/eLife.03582

    Figure Lengend Snippet: Genotype analysis of the generated PfΔ slarp and PfΔ b9 Δ slarp parasites . ( A ) Long range PCR analysis of genomic DNA from WT, Pf Δ slarp and Pf Δ b9 Δ slarp asexual parasites confirms the slarp gene deletion and consecutive gene deletions of both slarp and b9 respectively and subsequent removal of the hdhfr::gfp resistance marker. The PCR products are generated using primers P1,P2 for slarp and P3,P4 for b9 (see A and B respectively; for primer sequences see primer table in Supplementary file 2B ) and PCR products are also digested with restriction enzymes x ( Xma I) and kx ( Kpn I/ Xcm I) respectively for confirmation (i.e. slarp LR-PCR product sizes: WT, 12 kb, is undigested; Δ slarp-a , 5.4 kb is digested into 1.3 kb and 4.0 kb fragments, Δ slarp-b , 2.4 kb is digested into 1.3 kb and 1.1 kb fragments. b9 LR-PCR product sizes: WT, 5.5 kb, is digested into 756 bp, 793 bp, and 4.0 kb fragments; Δ b9-b , 2.6 kb is digested into 756 bp, 793 bp, and 1.1 kb fragments). ( B ) Southern analysis of restricted genomic DNA from WT, PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 asexual parasites. DNA was digested with restriction enzyme (E: Taq I) and probed with the 5′ slarp targeting region (P: 5′ slarp -T; see A ) on the left side of the slarp Southern or probed with the 3′ slarp targeting region (P: 3′ slarp -T; see A ) on the right side of the slarp panel. For analysis of the b9 , integration DNA was digested with restriction enzymes (E: Rca I) and probed with the 5′ b9 targeting region (P: 5′ b9 -T; see A ) on the right panel. The expected fragment sizes are indicated in panel ( A ). ( C ) RT-PCR analysis showing the absence of b9 and slarp transcripts in P. falciparum PfΔ slarp- a, PfΔ slarp- b, PfΔ b9 Δ slarp -F7, and PfΔ b9 Δ slarp -G9 mutant sporozoites. PCR amplification using purified sporozoite RNA was performed either in the presence or absence of reverse transcriptase (RT+ or RT−, respectively) and generated the expected 506 bp and 580 bp fragments for slarp and b9 respectively, the positive control was performed by PCR of 18S rRNA using primers 18Sf/18Sr (for primer sequences see Supplementary file 2B ) and generated the expected 130 bp fragment. DOI: http://dx.doi.org/10.7554/eLife.03582.009

    Article Snippet: All DNA fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen, Leek, The Netherlands) sub-cloning.

    Techniques: Generated, Polymerase Chain Reaction, Marker, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Amplification, Purification, Positive Control

    Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006

    Journal: eLife

    Article Title: A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites

    doi: 10.7554/eLife.03582

    Figure Lengend Snippet: Generation and genotype analyses of P. berghei mutant PbΔ slarp- a . ( A ) Generation of mutant PbΔ slarp -a. For PbΔ slarp -a, the DNA-construct pL1740 was generated containing the positive/negative selectable marker cassette hdhfr / yfcy . This construct was subsequently used to generate the mutant PbΔ slarp -a (1839cl3) in the Pb GFP-Luc con reference line. See Supplementary file 2A for the sequence of the primers. ( B ) Diagnostic PCR and Southern analysis of Pulse Field Gel (PFG)-separated chromosomes of mutant Δ slarp -a confirming correct disruption of the slarp -locus. See Supplementary file 2A for the sequence of the primers used for the selectable marker gene (SM); 5′-integration event (5′); 3′-integration event (3′); and the slarp ORF. Mutant PbΔ slarp -a has been generated in the reference P. berghei ANKA line Pb GFP-Luc con which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. For Southern analysis, PFG-separated chromosomes were hybridized using a 3′UTR pbdhfr probe that recognizes the construct integrated into P. berghei slarp locus on chromosome 9, the endogenous locus of dhfr/ts on chromosome 7, and the gfp-luciferase gene integrated into chromosome 3. In addition, the chromosomes were hybridized with the hdhfr probe recognizing the integrated construct into the slarp locus on chromosome 9. ( C ) Real time in vivo imaging of Δslarp luciferase-expressing liver-stage parasites in C57BL/6 mice at 24, 35, and 45 hr post-infection. C57BL/6 mice were IV injected with either 5 × 10 4 Pb -GFPLuc con sporozoites (n = 5), resulting in a full liver infection (upper panel: representative image of WT infected mice), or with 5 × 10 5 Pb Δslarp-a sporozoites (n = 5) (lower panel: representative image of Pb Δslarp-luc infected mice). DOI: http://dx.doi.org/10.7554/eLife.03582.006

    Article Snippet: All DNA fragments were amplified by PCR amplification (Phusion, Finnzymes) from genomic P. falciparum DNA (NF54 strain) and all PCR fragments were sequenced after TOPO TA (Invitrogen, Leek, The Netherlands) sub-cloning.

    Techniques: Mutagenesis, Construct, Generated, Marker, Sequencing, Diagnostic Assay, Polymerase Chain Reaction, Luciferase, In Vivo Imaging, Expressing, Mouse Assay, Infection, Injection