Summary

RMgm-931
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)
Phenotype Liver stage;
Last modified: 24 December 2014, 11:06
  *RMgm-931
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
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 cl15cy1
Other information parent line
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-931
Principal name1353cl2
Alternative nameΔsequestrin-b
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)

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.

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-930) was generated in a GFP-Luciferase expressing 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