Summary

RMgm-930
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1003000; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative | sequestrin | 6-cysteine protein (LISP2)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Liver stage;
Last modified: 24 December 2014, 11:08
  *RMgm-930
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24509910
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherT. Annoura; S.M. Khan; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-930
Principal name1424cl1
Alternative nameΔsequestrin-a
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageMutant sporozoites showed normal gliding motility and WT-levels of hepatocyte invasion. Mice infected with either 1 or 5x104 PbΔsequestrin sporozoites, intravenously, had a 2-3 day delay in blood-stage patency when compared to WT sporozoites infections and 4 out of 11 mice did not develop a blood-stage infection after inoculation with 1x104 sporozoites. These observations show that liver stage development is strongly affected in the absence of sequestrin and the 2-3 day prolonged prepatent period is indicative of a >99% reduction in liver-stage development. PbΔsequestrin liver stages have normal morphology, with respect to cell division, size and PVM formation at 24hpi. However at 48hpi, as determined by staining with anti-MSP1 antibodies, all liver-stage parasites were MSP1 negative. To investigate the maturation of these parasites, 54hpi parasites were examined using anti-MSP1 and anti-EXP1 antibodies. Over 60% WT parasites at this time point were strongly MSP1 positive, whereas the majority of PbΔsequestrin parasites were MSP1 negative, with only around 7% of parasites exhibiting similar MSP1 staining.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of sequestrin/lisp2 (liver stage specific protein 2; LISP2) and expresses the fusion gene GFP-Luciferase under the control of the constitutive eefa promoter

Protein (function)
PBANKA_100300 encodes a 2172 amino acid protein with a large repeat region of approximately 1000 amino acids. This region is composed of several repeat motifs, particularly 12 copies of a 56-amino acid motif. Analyses of the amino acid sequence with various structure analysis tools indicates that it has an N-terminal signal sequence but no other motifs for membrane association such as transmembrane domains or a GPI-anchor motif, suggesting that the encoded product has the structure of a secreted protein. It contains a predicted Plasmodium 6-cysteine motif. Several studies analysing expression of this protein in P. berghei (see 'Additional mutants') provide evidence for specific expression in liver stages. The protein has been named LISP2 and sequestrin.

Phenotype
Mutant sporozoites  showed normal gliding motility and WT-levels of hepatocyte invasion. Mice infected with either 1 or 5x104 PbΔsequestrin sporozoites, intravenously, had a 2-3 day delay in blood-stage patency when compared to WT sporozoites infections and 4 out of 11 mice did not develop a blood-stage infection after inoculation with 1x104 sporozoites.
These observations show that liver stage development is strongly affected in the absence of sequestrin and the 2-3 day prolonged prepatent period is indicative of a >99% reduction in liver-stage development.
PbΔsequestrin liver stages have normal morphology, with respect to cell division, size and PVM formation at 24hpi. However at 48hpi, as determined by staining with anti-MSP1 antibodies, all liver-stage parasites were MSP1 negative. To investigate the maturation of these parasites, 54hpi parasites were examined using anti-MSP1 and anti-EXP1 antibodies. Over 60% WT parasites at this time point were strongly MSP1 positive, whereas the majority of PbΔsequestrin parasites were MSP1 negative, with only around 7% of parasites exhibiting similar MSP1 staining.
Imaging of mice infected with 1x104 PbΔsequestrin sporozoites, showed a strong reduction in luminescence signals (>10-fold reduction) compared to signals found in mice infected with the same number of WT-luciferase expressing sporozoites.

Combined these observations show that sequestrin plays a role in late liver stage development which is in agreement with observations made by Orito et al. (RMgm-799) who show a role for sequestrin during late liver stage development and show that mutants lacking sequestrin have a 30-100 fold reduction in liver stage development.

Additional information

Supporting Figure S4
Generation of P. berghei mutants lacking expression of sequestrin (PbΔsequestrin)

A. Schematic representation of the construct pL1462 targeting sequestrin for gene deletion by double cross-over homologous recombination at the target regions (grey boxes), and the locus before and after disruption. The construct contains the tgdhfr-ts selection marker (SM). Two independent mutants were generated: Δsequestrin-a (1424cl1) and Δsequestrin-b (1353cl2). Primer positions for diagnostic PCRs and amplicon sizes are shown (see Table S1 for primer sequences). B. Diagnostic PCRs (left) and Southern analyses of separated chromosomes (right) confirm the correct integration of the constructs in Δsequestrin-a and Δsequestrin-b. Primer pairs and amplicon sizes are shown in A. Selectable marker (M; primers 4501/4502); 5’-integration event (5’; primers 4459/4703); 3’-integration event (3’; primers 4704/4460); ORF (O; primers 4472/4473). See Table S1 for the sequence of the primers. For Southern analyses, pulsed field gel-separated chromosomes were hybridized with a 3’UTR pbdhfr probe. The Δsequestrin-a mutant has been generated in the reference P. berghei ANKA PbGFP-Luccon which has a gfp-luciferase gene integrated into the silent 230p locus (PBANKA_030600) on chromosome 3. Hybridization with the 3’UTR dhfr/ts probe recognizes the integrated construct on chromosome 10, the reporter GFP-Luccon construct on chromosome 3, and the endogenous dhfr/ts gene located on chromosome 7. Δsequestrin-b has been generated in the reference P. berghei ANKA line cl15cy1. Hybridization with the 3’UTR dhfr/ts probe recognizes the integrated construct on chromosome 8 and the endogenous dhfr/ts gene located on chromosome 7. C. RT-PCR analysis showing absence of sequestrin transcripts in liver stages of Δsequestrin-a. PCR amplification using RNA of cultured liver stages (40hpi) was performed either in the presence or absence of reverse transcriptase (RT+ or RT-, respectively) using the primers as shown in the left panel (see Table S1 for the sequence of the primers). The P. berghei hsp70 gene was used as a positive control.


Other mutants
An identical mutant RMgm-(931) was generated in the WT background using the same targeting plasmid as described here. RMgm-799 describes an independent mutant lacking expression of Sequestrin/LISP2
RMgm-593 - A mutant expressing GFP under the control of the promoter of PBANKA_100300 (named lisp2 or sequestrin)
RMgm-800 - A mutant expressing a C-terminal tagged version of PBANKA_100300 (named lisp2 or sequestrin)

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1003000
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative | sequestrin | 6-cysteine protein
Gene product: Alternative nameLISP2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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AGCTTGCATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAAC
ATATAATTATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAA
AATATATAATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTT
TGTATGTTTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAG
ACACAAATAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATA
TTTTCTTTTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTT
ATTTTGTCATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAA
TAATTTTAACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTA
ATACACATTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATAT
ATATATATATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAA
TAATATATGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCA
TACATGCAATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATT
ATGGATATATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATT
TTATATATGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAAT
TTTTTTTTATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTG
AARGTTCGGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTT
TATACTTATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCC
AATTTTATAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTA
AGTGCTCAAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTA
CACATTAAAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAA
TTCTACAAATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTG
ATATATACATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATG
TATAGTATACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCC
TGTTTTTTATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATAT
ATAGGAAAAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACC
YTGGGTAGGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAA
AATTAACACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGT
TTAATATCAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATA
TTTATAATTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTC
AATTCGGGTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAA
AGRATTTGAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTA
TTTAGCAGCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATA
TAATACAATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGG
GTTATATAATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATT
AAGATATAAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAAT
TTTTATTTTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCA
TTTTAAAAATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTT
CATTTGTATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCAT
AAACCGGTGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGC
CTCCCGTGGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACG
CCCGAAGAAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGAC
TCTGGACTTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAA
ACCTGGGAAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTT
TCCTCTTCCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAG
CGCGTCCGAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAG
GATTCTGTCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCT
CTGGGCGTTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTT
TTCTTCCCTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCT
GCAGCTCCTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGAC
AATGAAGCGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCC
TACGACTTTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCG
AGCAACGCAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAG
GCTCCTTCTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGG
AAAAAACGCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCAT
GAAGAGTTCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGAT
GACCGAACGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGAT
CAGGCCTTTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTG
CTGTGGTTCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATC
TGGGACAAGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTC
GGAGACATCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGAC
ATGCACACAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTG
AGAACGAATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGAC
GAAATGGCGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAG
CTGTCGTGCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATC
GCTTCCTATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAG
TTCATTCACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAG
CAGCTGCGGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATC
AAGGAAATCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGA
CGAATCCAGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTT
TCTTACTTATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGT
GTGCATATTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTA
TTTTTTGTTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATAT
TTTATATAAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAA
CATAATATGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTAT
AATTATGTTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATA
ATATGATTAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACA
AATGATGTTTTTTCCTTCAATTTCGATATCGAATTCGAAGAAAACAATAAAGAGCTACCA
AAATTTAAAGAAAATCCTAAAAGAGGTAATACCAAAAAATATGCATATAAGAAAAAGCCC
AAACCATATTGGAAATACATGATATAAGGAATGAAATTCAAAGTAAAAAAGAAAAATGCA
CTATTAATTTGGAAGTATTGAACGTGATAACAAAAACAAAACAAAGGAGTGAAATCAAAA
GAAAATAAGACGGAAAGTCCGAAAGAAGTAATAATTCAAATAAATGTAATAATATAAAAT
ACCTAAAGTTTAGCTATATAAATAAGTATTCTAATTAAGTATCTTATAATAATATATTTT
TTTTTCTATACTATTATAAATAAAAATGTTAGCATATATGTGTCTTTGTGTAATTTTATA
TTCAGATTCAAAGCATTTACCATTTATGATTATTTTACTAAGGTTGTCCTAATTTAAATT
ATAATTAGAAAAAACTAGTATATATTTGAATAGAATTTTAATTACATATTATAAAAAAAA
CATAATTTTTTATTATAAAATATTTCATTTCATTTTATTAACCTACAAGGCATTATTTAA
CATTAACTTCCTCAAAAAATGTAAAAAATAAATTAAAAAAATTTTCTTATTCCATTTTTT
TTATTTTTATGAGATTTGTGCAAGAACAAAATATTTTATTAAAATTTTTAAAAAAATAAA
TACATAAAATTTAAAGATTTCATAAATGTTTTTAAAAGAAAATAAAAATAAGAAGCATAA
AGCATATAATATTTATTTTTCTTGAGTAATGGTATAGATTATTTGGTATCTATATATAAT
TTGATGATATAAATACTGTAAAAGGCAATAATTTAATTTCTCATAGTTTAATAATGACTA
ATTATATTGATTAATTCATATGTATTATGTTTGGTTGCTTAGACTATTGTGATATCAAGT
CTTTCCATTTTCACTAATTATTTGACATTTTTTATTTCATCTAACGATATTTTGTAGTTT
TACTTGAAGTTATTTCCTTTATATATATAATTTGATTAATAAATATGTTTATTTTCCATC
ATTTAATTTCTTAACTCGAATTTCTTCGAATAATTAATCATTTAGAGTTTCTTGTTTTAT
TCCTGGTTCTTTGTTCCACATTGCTTCTATTAGTGCATCCGTGGATGTCCCACTTAGTAT
ATCTATTTCTTTGTCTAATTTTGATTCGACGATTTCATCTTTCAAAGATTCGTTCGCTGT
ATCTACCATACTAGTAGAACAATTTTCTAGTAATATATCATTTAGGAAATTGTTTTTTCT
CTTTGGGATATCTTCGTAACTATTTTTTTCGGCATACATTTCTTTAATCATTTTGTGGGT
ACTTTTCGGTAAGCTACGTCGATATCGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCG
GTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACA
ACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC
TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCG
CAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGT
GGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTT
CTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCT
CCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG
TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGA
GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTC
GGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGA
GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGT
GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA
AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGG
AAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGC
CTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTG
GGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTT
CGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTA
TTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAAT
GACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA
GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA
ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACT
CGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC
ACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACT
CTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTT
CTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGT
GGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTT
ATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATA
GGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAG
ATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT
CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAA
AAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA
AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT
CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG
TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC
CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGA
CGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCC
AGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC
GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACA
GGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG
TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTA
TGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT
CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAG
TGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA
GCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGC
AGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTG
AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTG
TGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCC
AAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAGCTGGTACCGACCTTCTAGATTAAT
TGCAGAAAAGGAATCTGAAAATAATAGCGAAAATAATAGTCAAGATGTAAAACAAGATAA
AGAATCTAATACTTCTGATAAGAACAGTGATGATTCAAATCATGATAATCCCGATTCACC
AGTTCAAAATGATATACCAGAGGGCAACATTAAAAGACCTTCTAGATTAATTGCAGAAAA
GGAATCTGAAAATAATAGCGAAAATAATAGCGAAAATAATAGCGAAAATAATAGTCAAGA
TGTAAAACAAGATAAAGAATCTAATACTTCTGATAAGAACAGTGATGATTCAAATCATGA
TAATCCCGATTCACCAGTTCAAAATGATATACCAGAGGGCAACATTAAAAGACCTTCTAG
ATTAATTGCAGAAAAGGAATCTGAAAATAATAGCGAAAATAATAGTCAAGATGTAAAACA
AGATAAAGAATCTAATACTTCTGATAAGAACAGTGATGATTCAAATCATGATAATCCCGA
TTCACCAGTTCAAAATGATATACCAGAGGGCAACATTAAAAGACCTTCTAGATTAATTGC
AGAAAAGGAATCTGAAAATAATAGCGAAAATAATAGCGAAAATAATAGCGAAAATAATAG
CGAAAATAATAGCGAAAATAATAGCGAAAATAATAGTCAAGATGTAAAACAAGATAAAGA
ATCTAATACTTCTGATAAGAACAGTGATGATTCAAATCATGATAATCCCGATTCACCAGT
TCAAAATGATATACCAGAGGGCAACATTAAAAGACCTTCTAGATTAATTGCAGAAAAGGA
ATCTGAAAATAATAGCGAAAATAATAGCGAAAATAATAGTCAAGATGTAAAACAAGATAA
AGAATCTAATACTTCTGATAAGAACAGTGATGATTCAAATCATGATAATCCCGATTCACC
AGTTCAAAATGATATACCAGAGGGCAACATTAAAAGA
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1GGGGTACCCAACAGCAATATATCGTCACC
Additional information primer 14261 (Asp718); ∆PBANKA_100300 5' target F
Sequence Primer 2CCCAAGCTTCGTGTTTCCTTTCTTTTTCTCG
Additional information primer 24262 (Asp718); ∆PBANKA_100300 5' target R
Sequence Primer 3GGAATTCGAAGAAAACAATAAAGAGCTACC
Additional information primer 34263 (Asp718); ∆PBANKA_100300 3' target F
Sequence Primer 4CGGGATCCCGATATCGACGTAGCTTACCG
Additional information primer 44264 (Asp718); ∆PBANKA_100300 3' target R
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
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Plasmid/construct sequence
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1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
61 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
121 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
181 ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc
241 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
301 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
361 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
421 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
541 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
601 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
661 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
721 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
781 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
841 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
901 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
961 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
1021 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
1081 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
1141 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
1201 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
1261 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
1321 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
1381 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
1441 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
1501 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
1561 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
1621 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
1681 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
1741 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
1801 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
1861 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
1921 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
1981 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
2041 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
2101 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
2161 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
2221 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
2281 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
2341 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
2401 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
2461 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
2521 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
2581 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt
2641 ccgcgggtat atggtaaaga acctactaac acaataaaat atttaaataa tgtatttcct
2701 ataaataaat ttacagattt attttttaat acaaaagata tagatatacc agaaataaat
2761 gatcagttta aaggttttaa attctttatg acatcattta taaatcatgg atcatatcca
2821 ctaacaatag aatgtggtgt aacaaatggt ggaactagtt ataaaagagc aattatttta
2881 ttgcatgttc gaactgattt aaaagataga ccagtttcat tttgtgattt tcgaaaagga
2941 gaattatata attatttgaa tgcttatact gaaggggatg tatgcataat aatttccaaa
3001 tcaaatacaa gttttggttt tagatgccca gtaaatacaa aaaaaatgcc aaaaaattgt
3061 tttacgcaag tatatgaaaa agggtatcta aatgacgcca ataaaattaa tactaaaaat
3121 gttattaact attcatttga aaatccagaa tatgcgctag ctggttytaa ttatacatta
3181 acaaaatcgt atcaatttga atgtcattgt gtagataaag aaacagaaca aattgtaaaa
3241 acggttttag tcaaatatgt aaatgaagat gaaatatatg attataatga ttttccaatg
3301 gtgaatcaca aacctattat tgcacatcca aataaaacac atcaagcttg catgcctgca
3361 gcccagctta attcttttcg agctctttat gcttaagttt acaatttaat attcatactt
3421 taagtatttt ttgtagtatc ctagatattg tgctttaaat gctcacccct caaagcacca
3481 gtaatatttt catccactga aataccatta aattttcaaa aaaatactat gcatataatg
3541 ttatacatat aaacataaaa cgccatgtaa atcaaaaaat atataaaaat atgtataaaa
3601 ataaatatgc actaaatata agctaattat gcataaaaat taaagtgccc tttattaact
3661 agtcgtaatt atttatattt ctatgttata aaaaaatcct catataataa tataattaat
3721 atatgtaatg ttttttttat tttataattt taatataaaa taatatgtaa attaattcaa
3781 aaaataaata taattgttgt gaaacaaaaa acgtaatttt ttcatttgcc ttcaaaattt
3841 aaatttattt taatatttcc taaaatatat atactttgtg tataaatata taaaaatata
3901 tatttgctta taaataaata aaaaatttta taaaacatag ggggatctat gagtaaagga
3961 gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga tgttaatggg
4021 cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa acttaccctt
4081 aaatttattt gcactactgg aaaactacct gttccatggc caacacttgt cactactttc
4141 ggttatggtg ttcaatgctt tgcgagatac ccagatcata tgaaacagca tgactttttc
4201 aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa agatgacggg
4261 aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa tagaatcgag
4321 ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt ggaatacaac
4381 tataactcac acaatgtata catcatggca gacaaacaaa agaatggaat caaagttaac
4441 ttcaaaatta gacacaacat tgaagatgga agcgttcaac tagcagacca ttatcaacaa
4501 aatactccaa ttggcgatgg ccctgtcctt ttaccagaca accattacct gtccacacaa
4561 tctgcccttt cgaaagatcc caacgaaaag agagaccaca tggtccttct tgagtttgta
4621 acagctgctg ggattacaca tggcatggat gaactataca aagggatcct ggctagccag
4681 tcgacctgca ggcatgcaag cttgcggccg atccaaatgg aagacgccaa aaacataaag
4741 aaaggcccgg cgccattcta tccgctggaa gatggaaccg ctggagagca actgcataag
4801 gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc acatatcgag
4861 gtgaacatca cgtacgcgga atacttcgaa atgtccgttc ggttggcaga agctatgaaa
4921 cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca gtgaaaactc tcttcaattc
4981 tttatgccgg tgttgggcgc gttatttatc ggagttgcag ttgcgcccgc gaacgacatt
5041 tataatgaac gtgaattgct caacagtatg aacatttcgc agcctaccgt agtgtttgtt
5101 tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa aattaccaat aatccagaaa
5161 attattatca tggattctaa aacggattac cagggatttc agtcgatgta cacgttcgtc
5221 acatctcatc tacctcccgg ttttaatgaa tacgattttg taccagagtc ctttgatcgt
5281 gacaaaacaa ttgcactgat aatgaattcc tctggatcta ctgggttacc taagggtgtg
5341 gcccttccgc atagaactgc ctgcgtcaga ttctcgcatg ccagagatcc tatttttggc
5401 aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca cggttttgga
5461 atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat gtatagattt
5521 gaagaagagc tgtttttacg atcccttcag gattacaaaa ttcaaagtgc gttgctagta
5581 ccaaccctat tttcattctt cgccaaaagc actctgattg acaaatacga tttatctaat
5641 ttacacgaaa ttgcttctgg gggcgcacct ctttcgaaag aagtcgggga agcggttgca
5701 aaacgcttcc atcttccagg gatacgacaa ggatatgggc tcactgagac tacatcagct
5761 attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt tgttccattt
5821 tttgaagcga aggttgtgga tctggatacc gggaaaacgc tgggcgttaa tcagagaggc
5881 gaattatgtg tcagaggacc tatgattatg tccggttatg taaacaatcc ggaagcgacc
5941 aacgccttga ttgacaagga tggatggcta cattctggag acatagctta ctgggacgaa
6001 gacgaacact tcttcatagt tgaccgcttg aagtctttaa ttaaatacaa aggatatcag
6061 gtggcccccg ctgaattgga atcgatattg ttacaacacc ccaacatctt cgacgcgggc
6121 gtggcaggtc ttcccgacga tgacgccggt gaacttcccg ccgccgttgt tgttttggag
6181 cacggaaaga cgatgacgga aaaagagatc gtggattacg tcgccagtca agtaacaacc
6241 gcgaaaaagt tgcgcggagg agttgtgttt gtggacgaag taccgaaagg tcttaccgga
6301 aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg cggaaagatc
6361 gccgtgtaat tctagaagat cccgtttttc ttacttatat atttatacca attgattgta
6421 tttataactg taaaaatgtg tatgttgtgt gcatattttt ttttgtgcat gcacatgcat
6481 gtaaatagct aaaattatga acattttatt ttttgttcag aaaaaaaaaa ctttacacac
6541 ataaaatggc tagtatgaat agccatattt tatataaatt aaatcctatg aatttatgac
6601 catattaaaa atttagatat ttatggaaca taatatgttt gaaacaataa gacaaaatta
6661 ttattattat tattattttt actgttataa ttatgttgtc tcttcaatga ttcataaata
6721 gttggacttg atttttaaaa tgtttataat atgattagca tagttaaata aaaaaagttg
6781 aaaaattaaa aaaaaacata taaacacaaa tgatgttttt tccttcaatt tcgggtaccg
6841 agctcgaatt ctcttgagcc cgttaatgaa atagatacaa ttcattcatg ttatatacat
6901 ctagaacata atctgaatat ggttcaagtt aaatgtccaa aaattataaa aagtgatgat
6961 atttttgatg gtaataccat aatagacacc aaggtaacat cacgaagtag tcaacaaaat
7021 aatttttatt tagaaaatac agatgttgaa ccagaagaaa tagagaaata taaaaatata
7081 gaatacatac cagaaaacga tgaagtaatg catctagaca aaaaagaaaa gctagatgat
7141 atattaccag gtgttatcat attagataaa aataaaatgt tcaaagaaaa aggacatttc
7201 acttttgtta ctccattaat tgtagaaaag gtattaatat taaaaatata ttgtgataat
7261 actaaaacaa taattaataa tatgaaaggg aaaaaaggta ttacagtaat aaggatttct
7321 caaaatacaa caaaaaataa attttatgga tgtgactttt caggtaattc taaaaaaaca
7381 ttttactatt ccaatgttta tgatttagaa aaaaaaaatg agttttgtga aatagaatta
7441 aaagaaaata tagtagttag cttaaattgt ccaactggta aaattaatcc aaaaaattgt
7501 tttagaaatg tatatataaa aagtaatatg aatgaacaaa caaccgaaaa tatagaaaat
7561 atatttaacg aaataaaagt tatagatgca gattatttta taaataattc atcaaccttt
7621 ttgatgattt ccaaaattac aaaaaaagag tttgattttt attgtacatg tgaagattat
7681 aaaaccaaaa atataggaac aatatatatt aaaaattatg aatatctaga ttcaaaacct
7741 aaatataaaa ataaacaaat ttcctatata gatgtagttc catacccgcg gggaaagggc
7801 g
Restriction sites to linearize plasmid ApaI, SacII
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4