RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-124

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1300700; Gene model (P.falciparum): PF3D7_1475500; Gene product: LCCL domain-containing protein (LAP2; LCCL/lectin adhesive-like protein 2; CCP1)
Disrupted	Gene model (rodent): PBANKA_1035200; Gene model (P.falciparum): PF3D7_1407000; Gene product: LCCL domain-containing protein \| scavenger receptor-like protein (PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3)
Phenotype	Oocyst; Sporozoite;

Last modified: 13 January 2011, 08:49

*RMgm-124

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene disruption, Gene disruption
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 18848846
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/Researcher	C. Lavazec, G.R. Mair, C.J. Janse, J. Templeton
Name Group/Department	Department of Microbiology and Immunology
Name Institute	Weill Cornell Medical College
City	New York
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-124
Principal name	∆PbCCp1/∆PbCCp3
Alternative name	952 m1-m0cl1
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Not different from wild type
Oocyst	Normal numbers of oocysts are produced. Sporozoite formation within the oocysts is blocked. Analysis of the morphology of oocysts by light microscopy revealed that the majority of 17–22 day oocysts were either vacuolated or non-sporulated, and did not contain sporozoites.
Sporozoite	No hemocoel and salivary gland sporozoites were detected.
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of two proteins CCp1 (LAP2, LCCL/lectin adhesive-like protein 2; LCCL domain containing protein CCp1) and CCp3 (PbSR, P. berghei Scavenger Receptor-like protein; PSLAP; LAP1). Protein (function) The ccp1 and ccp3 genes are members of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's and in P. berghei PbLAP's. Phenotype Phenotype analyses indicate a role during oocyst development and sporozoite formation/production (see also additional information). Additional information In another study (Raine, J.D. et al., 2007, PloS Pathogens 3, e30) it has been shown that crossings of ∆lap2 (ccp1) and ∆lap1 (pbsr/ccp3) mutant females with wild type males did not rescue the formation of sporozoites. In contrast, crossing of the mutant males with wild type females resulted in wild type production of sporozoites that were infectious to C57BL/6 mice. The lack of rescue of the mutant phenotypes by crossing of mutant females with wild type males is suggestive of a role of these proteins within a few hours after fertilisation. Other members of the LAP/CCp family of proteins have been analysed by targeted gene disruption. A number of mutants lacking expression of these proteins show a comparable defect in development of oocysts and formation of sporozoites (RMgm-113, RMgm-114, RMgm-115: LAP1/PbSLAP/PbSR/CCp3; RMgm-119: LAP4/CCp2; RMgm-98: LAP5/FNPA; RMgm-120: LAP6, CCp4). Other mutants RMgm-118: A mutant lacking expression of LAP2/CCp1 RMgm-121: A mutant lacking expression of LAP2/CCp1 RMgm-113: A mutant lacking expression of PbSR/PSLAP/LAP1/CCp3 RMgm-114: A mutant lacking expression of PbSR/PSLAP/LAP1/CCp3 RMgm-115: A mutant lacking expression of a mutated form of PbSR/PSLAP/LAP1/CCp3 RMgm-125: A mutant lacking expression of both LAP2/CCP1 and LAP6/CCp4

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1300700

Gene Model P. falciparum ortholog PF3D7_1475500

Gene product LCCL domain-containing protein

Gene product: Alternative name LAP2; LCCL/lectin adhesive-like protein 2; CCP1

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AATTCGGAATTACTGAATAATGAAAAGCAAATAATAATATAAATAACAAATCATAATAAT

AGAATATATTATACAATGTGAAGTAAAATGATTATTTTTGAACGAAAATTATTTATTTGT

TCATTTGAACATATGCTTATGTGATGTTTTTTCTTATTTAGGTTGATTGGTTCACCAATC

ATGATAATTTTTTTATTGATGACACGTGGAAAGACAATAAATTAAATTTAAAACATATGC

AAAGCAATTTATTTCTATTACACATATACAGCATTATTATTATATGCTTTGTTTTCTCCT

AAATTAGCAAACTGTTTAGAAACAAATATAGGAAAAATCTATATATGCGCATATCCTTAA

TAAACAGTAGAATTATATAAATATAATAATATGCATACATTATTTGGTTAGTTCATTTTG

CGTATAAAAGCAATATATCGTGATCGTCTTTTGGGTTTTTTTTATCATGTAAAAAATACG

GGTTATTGTCCCCTAAATCAATTTTGCTAAATATAATAAAATCATATTAAGGGCGCACTT

TAGTTAATTATAACAAAATAAATGCATATATAGTGTAATGTTGATAGGATTATTTATAAT

TTTTTTAGATTCATGGTTCGTTTTGCTAATTTTATGAAATTTTCTTTGAGGTATTTATGT

ATGAACTTATTTAAATATTTGTATTATAGCAGTGAAATATATATTCCATCCATATAATAA

AATAAATAATTTATTGTATAAAATTGTAAATAGTCTTCTTATATATATAATAAATATAAT

TTTTATTAAATTTTATTATAAAAAGGGTTCAATTTCTTTATGAAAGTTTAAAATTTATAA

TAAAAAAGTACAAATAATTTTTATTTTGCAATAAGGAGCTGCATAAAGAAAAAACATAGA

TATCCAAAATGTGTAATAAAAGATATTTTTAGTGCGTATAATATACATACATTAACTAGT

TCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGC

GCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACT

TAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCAC

CGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGG

CGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGC

CCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCC

CCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT

CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGAC

GGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAAC

TGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGAT

TTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAA

AATATTAACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATT

TGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT

ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAA

GTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC

AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTT

AAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT

CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCAT

CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAAC

ACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG

CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCC

ATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAA

CTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAG

GCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCT

GATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGAT

GGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAA

CGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGAC

CAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTC

CACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTG

CGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCG

GATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCA

AATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCG

CCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGA

ACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATAC

CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT

CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCC

TGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGA

TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTC

CTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTG

GATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAG

CGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCC

GCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGC

AGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACAC

TTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGA

AACAGCTATGACCATGATTACGCCAAGCTCGAAATTAACCCTCACTAAAGGGAACAAAAG

CTGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTATTAAAATTAATACGAA

AATAAAAATTAAAGGATGATGGCGATTATTAGTTCTTTTTAATTCCATCATCTATCCTTA

TATGTAGTATATTGGGGTTAGAGATAGTGGGTTATGAATACCAAAAATATAGAAAATTGC

AAAGGAGTTGATTTATTCTCTGTTGTATAAGAATGTACACAGTTAAAGTTAGTATATATG

TGTATATTTTTCAGTCTATATAAATATACTTTTGTTTTCTCATTGGTATGTTCAATTTTT

TATATTATTTTACGTGTCGATGGATACAATTTAGTATTTTTTTTAATTTTGTATTTTTGA

ATTATATTAATCTAGATTATTCATATATTTTTTCATATGCAGATAGAAAAAGGTTTCATA

CGTTAGTTTATCCTACAAATTGAATATTAACACATACATATATTTATATTTTAAGCTTGC

ATGCCTGCAGGTCAACAATAAATAATAAATAAATATTGTGGAAATAAAATAACATATAAT

TATTTTTAATACATTGATTTCCCTTTTATTTTTTTAAATTTCATTGATATAAAAATATAT

AATAATAACATATATGATTTCAAATTAATCTTTTCAAAAATGGTGTTTATTTTTGTATGT

TTGTGTATGAATTAATCACATAACACATCTATTAAATTGAGTTGGTAATATAGACACAAA

TAAATATATATATTTTTATAGCTTAAAAGTGTGTTATGAATATTTTAAGCATATTTTCTT

TTTCTTTGGATTGTGTAAAATGAACTCATATAATGCGTTTTTTTGTTTTTGTTATTTTGT

CATTTTGTTATTTTGCTATTTTATGGATTAATTTTTGTTTATAAAATGGGAAATAATTTT

AACATATTTAAATAAATGGAGAAAAAATATAAAATAATTATAAAAAAAAGTTAATACACA

TTTTTTCCTGTTATAGACCTTATATTTATTTATCCATATATATATATATATATATATATA

TATATATATACATACCAAGTGAATTAAGAGGAAAGCTAATTTATTATTCAGAATAATATA

TGAACTATATATAATTTTTATTATTTTGGTGTATATTAATCTGTCTATATGCATACATGC

AATAATTTATCGACTTATATATCAAATAACATAAAATAGAAGTGTTTTAAATTATGGATA

TATGCTCAATATTCATTTTTTTTAATAAGTTAGCTATATTTAAATTATACATTTTATATA

TGGTCTCTTTTTTTTTTAAATATTATTTAAGTGATCATGAAAATATAAATAATTTTTTTT

TATTTAATATCCTTTTGCTTGCATGTGGTAAATGGAAATTTGGATGTGTTTTGAARGTTC

GGATATAGTTGTATGGACATATAATATATTTTGTGAAAAATTGGTTTTATGTTTATACTT

ATGCCAATACTTTTTGAGTAAAACAAAGCAAGTGCTTATAAATAATTAAAGCCAATTTTA

TAATATATATTTTTTTATTTAATTTGAATTTAGTAGTATAATTTTTTATGGTAAGTGCTC

AAAGAGAGTTGCTTATAAAGTATGGTTTGTTTCTTTTTCGCCATTTTGAATTACACATTA

AAAATATATAGATACATATATTATAATATGAAATCATTAATAATTTAGGGAAATTCTACA

AATTTAAAAACGAATAAAATAATTGTTTTTCATCATGCCATAACACAATATTGATATATA

CATGTACAAACATTTTTTTTATTTGGAAAATATAAATTATATAAAAAAAAATGTATAGTA

TACAAAATGAGCATATTCACACGGGGTGGACGTTCATTTTTTCATTTTTCCCCTGTTTTT

TATGAGTATATGATAAAATTTTATGAACATTTACACAAAATGAAAATGGATATATAGGAA

AAATGGAGCGGTATTTCATTTATCTTTGATTGTCATTTGGATATTATATTACCYTGGGTA

GGCAATTAAAAATGTTAAATAACAATTTAAGGAAATTATATTTTATATATTAAAATTAAC

ACTGTATTATATGATTCGCTTATAAAAGCCACTCTTTCCCCATGCAAAGCTGTTTAATAT

CAATTTTAACAAATTACACACATGTTAATATATTTATATATATAATTTATATATTTATAA

TTTATATATTTATATTTTTATTATTTATATATTTATTATTTATTGTGTGTGTCAATTCGG

GTAGGATATACCTCTTTTTTATTGTTTAAAGCGATTTGTATTCTAAAATATAAAGRATTT

GAAAAAGAGAAAGATAGAATATGATCCCATCATATATAGCCCTATAATTTTTATTTAGCA

GCGAATTAATTTTTCTATTAAGTTTATGTGTAATTAAAATAACGGAATATATATAATACA

ATAAAAAAGTGCATAAATTAAAATTTTTTCAATTAAATTTTTTTTTTTAAGGGGTTATAT

AATATTAAATATATAAAATACGATTATATATTTTTGCTACAATTTTTTATATTAAGATAT

AAATAGTAAATAAATGGTATTATATGGCATGTAATATATAAATTTTTTCCAATTTTTATT

TTATATACACTTTTCCTTTTTTTGTCATAAAACTTAAACAATTTACACATTCATTTTAAA

AATTGACTATTTGTTTCAACATTTTTTGAGTTTCCGTTTTATAATAGTATTTTCATTTGT

ATATTGCTTATATATATAAATACACACCTAAATGTTACAAAGGATCAATGCATAAACCGG

TGTGTCTGGTCGTCGCGATGACCCCCAAGAGGGGCATCGGCATCAACAACGGCCTCCCGT

GGCCCCACTTGACCACAGATTTCAAACACTTTTCTCGTGTGACAAAAACGACGCCCGAAG

AAGCCAGTCGCCTGAACGGGTGGCTTCCCAGGAAATTTGCAAAGACGGGCGACTCTGGAC

TTCCCTCTCCATCAGTCGGCAAGAGATTCAACGCCGTTGTCATGGGACGGAAAACCTGGG

AAAGCATGCCTCGAAAGTTTAGACCCCTCGTGGACAGATTGAACATCGTCGTTTCCTCTT

CCCTCAAAGAAGAAGACATTGCGGCGGAGAAGCCTCAAGCTGAAGGCCAGCAGCGCGTCC

GAGTCTGTGCTTCACTCCCAGCAGCTCTCAGCCTTCTGGAGGAAGAGTACAAGGATTCTG

TCGACCAGATTTTTGTCGTGGGAGGAGCGGGACTGTACGAGGCAGCGCTGTCTCTGGGCG

TTGCCTCTCACCTGTACATCACGCGTGTAGCCCGCGAGTTTCCGTGCGACGTTTTCTTCC

CTGCGTTCCCCGGAGATGACATTCTTTCAAACAAATCAACTGCTGCGCAGGCTGCAGCTC

CTGCCGAGTCTGTGTTCGTTCCCTTTTGTCCGGAGCTCGGAAGAGAGAAGGACAATGAAG

CGACGTATCGACCCATCTTCATTTCCAAGACCTTCTCAGACAACGGGGTTCCCTACGACT

TTGTGGTTCTCGAGAAGAGAAGGAAGACTGACGACGCAGCCACTGCGGAACCGAGCAACG

CAATGAGCTCCTTGACGTCCACGAGGGAGACAACTCCCGTGCACGGGTTGCAGGCTCCTT

CTTCGGCCGCAGCCATTGCCCCGGTGTTGGCGTGGATGGACGAAGAAGACCGGAAAAAAC

GCGAGCAAAAGGAACTGATTCGGGCCGTTCCGCATGTTCACTTTAGAGGCCATGAAGAGT

TCCAGTACCTTGATCTCATTGCCGACATTATTAACAATGGAAGGACAATGGATGACCGAA

CGGGCGTTGGTGTCATCTCCAAATTCGGCTGCACTATGCGCTACTCGCTGGATCAGGCCT

TTCCACTTCTCACCACAAAGCGTGTGTTCTGGAAAGGGGTCCTCGAAGAGTTGCTGTGGT

TCATTCGCGGCGACACGAACGCAAACCATCTTTCTGAGAAGGGCGTGAAGATCTGGGACA

AGAATGTGACACGCGAGTTCCTCGATTCGCGCAATCTCCCCCACCGAGAGGTCGGAGACA

TCGGCCCGGGCTACGGCTTCCAGTGGAGACACTTCGGCGCGGCATACAAAGACATGCACA

CAGACTACACAGGGCAGGGCGTCGACCAGCTGAAGAATGTGATCCAGATGCTGAGAACGA

ATCCAACAGATCGTCGCATGCTCATGACTGCCTGGAATCCTGCAGCGCTGGACGAAATGG

CGCTGCCGCCTTGTCACTTGTTGTGCCAGTTCTACGTGAACGACCAGAAGGAGCTGTCGT

GCATCATGTATCAGCGGTCGTGCGATGTCGGCCTCGGCGTCCCCTTCAACATCGCTTCCT

ATTCGCTTTTGACGCTCATGGTTGCACACGTCTGCAACCTAAAACCTAAGGAGTTCATTC

ACTTCATGGGGAACACGCATGTCTACACGAACCATGTCGAGGCTTTAAAAGAGCAGCTGC

GGAGAGAACCGAGACCGTTCCCCATTGTGAACATCCTCAACAAGGAACGCATCAAGGAAA

TCGACGATTTCACCGCCGAGGATTTTGAGGTCGTGGGCTACGTCCCGCACGGACGAATCC

AGATGGAGATGGCTGTCTAGCGGAAATACAGAAGCTAGCTTTGATCCCGTTTTTCTTACT

TATATATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATA

TTTTTTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTG

TTCAGAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATAT

AAATTAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATA

TGTTTGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATG

TTGTCTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGAT

TAGCATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATG

TTTTTTCCTTCAATTTCGATATCG

Restriction sites to linearize plasmid ClaI/SpeI double digest

Partial or complete disruption of the gene Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite tgdhfr

Promoter of the selectable marker pbdhfr

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification The ccp1/lap2 gene has been disrupted using a construct that contains the tgdhfr selectable marker and that integrates by double cross-over recombination. This mutant line (429cl1) has been described as mutant RMgm-121. The ccp3 (pbsr, lap1) gene has been disrupted in this line.
The ccp3 gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the hdhfr selectable marker. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.

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 1	5'-AAATCGATTAAAGCTATTAAAATTAATACG
Additional information primer 1	primer 1795
Sequence Primer 2	5'-TTAAGCTTAAAATATAAATATATGTATGTG
Additional information primer 2	primer 1796
Sequence Primer 3	3'-AAGAATTCGGAATTACTGAATAATGAAAAGC
Additional information primer 3	primer 1797
Sequence Primer 4	3'-TTACTAGTTAATGTATGTATATTATACGC
Additional information primer 4	primer 1798
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_1035200

Gene Model P. falciparum ortholog PF3D7_1407000

Gene product LCCL domain-containing protein | scavenger receptor-like protein

Gene product: Alternative name PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used Plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT

AATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC

GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT

CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC

TCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA

CGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC

ATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA

CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACT

TTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATG

TATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT

ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCT

GTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCA

CGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCC

GAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCC

CGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG

GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTA

TGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATC

GGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATG

CCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT

TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGC

TCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCT

CGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC

ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC

TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGAT

TTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATG

ACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATC

AAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA

CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAG

GTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTA

GGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTA

CCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAG

TTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG

GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACG

CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAG

CGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC

CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

AACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATG

TTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCT

GATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAA

GAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGG

CACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAG

CTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGA

ATTGTGAGCGGTAAACAATTTCACACAGGAAACAGCTTGACCATGATTACGCCAAGCTTA

TTTGTTCAGAAGGGTGGACAAAAGAAACAGAAAAAATTGCATGTTTAGAATTAGGTTATC

ATAATGTAAAAGCAAATGGATTCTCTCATCATCTGTGTAGTGATATAGCTGGGGAAAATT

TATGTGGTCATGATAAAGAAAGAATTAATGCAACTAATTTTAGATGTAAAGGAGATGAAG

CTAATTTAAAAAATTGCCCACATGAAACATCTGAAGATATTTATTGCTCTCACGAAGAAG

ATATAATTATTGGTTGCGCAAGTGCAGAAGAGGAAGGAAATGATTCTGCCAACTCAACTA

ACATTTCTAAATATGGATTAAACAAACATATGATGTCTATGGAAAAAAAAAAATTTCATC

CAAAAATCGAATTAAGTTGCTTTGACAAAATAAGCTCTAAGGCTGAGTTAAGCAAAGGAA

ATGTTGGAGATATATTTTTAGTTAGTTGTCCAGAGAAATGTGACGAAGATATTGGTGTAA

TAAAAGGAACGTTTGTATATACATTCGATTCTTATATATGTAAAGCAGGGATCCATGCAG

GTGTTTTGTCGAGTAGCGTTACTGATGATATGATCCTTATCATTACCCATAGTAGAAATA

AATTTATAGGAACGAAAAGAAATAATATAGAATCAAAAGAATTTAATGGAGAATCTAAAA

GTTTCAGCTTAAGTATACCAACAAATTATATCATAATGGAAGAAAGACAAAATAATTCAA

AATATGAAGATGAAATTTTAAAAGAAGATAACGATTTTTATTATGAGCATATATTTAAAA

AACAAAATAATAATGAACAAGAGAAAAATAAAACATTTTTCGAGCATAATATGCATCTAG

AACCAACTTTTCAATGGCTAGCTCCATCTTCATTTGCTGGATTCAATGGAGATGAAAATC

AATATATAAATGCTAATAATCTTCCTAACAAAAAATATATTAGAACTCTTTCTAATTTTA

CTTTTATTATCCATTTTATTCCAAATAGTAAAGGAAAAAATAAATGGAGAACAATATTAT

CTCATAGTTTGTGTGAAGGACTGCAGCCCAGCTTAATTCTTTTCGAGCTCTTTATGCTTA

AGTTTACAATTTAATATTCATACTTTAAGTATTTTTTGTAGTATCCTAGATATTGTGCTT

TAAATGCTCACCCCTCAAAGCACCAGTAATATTTTCATCCACTGAAATACCATTAAATTT

TCAAAAAAATACTATGCATATAATGTTATACATATAAACATAAAACGCCATGTAAATCAA

AAAATATATAAAAATATGTATAAAAATAAATATGCACTAAATATAAGCTAATTATGCATA

AAAATTAAAGTGCCCTTTATTAACTAGTCGTAATTATTTATATTTCTATGTTATAAAAAA

ATCCTCATATAATAATATAATTAATATATGTAATGTTTTTTTTATTTTATAATTTTAATA

TAAAATAATATGTAAATTAATTCAAAAAATAAATATAATTGTTGTGAAACAAAAAACGTA

ATTTTTTCATTTGCCTTCAAAATTTAAATTTATTTTAATATTTCCTAAAATATATATACT

TTGTGTATAAATATATAAAAATATATATTTGCTTATAAATAAATAAAAAATTTTATAAAA

CATAGGGGGATCCATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGG

CATCGGCAAGAACGGGGACCTGCCCTGGCCACCGCTCAGGAACGAATTTAGATATTTCCA

GAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAA

GACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCT

CAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGA

TGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGT

TGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGT

GACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAA

ATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCAT

TAAGTACAAATTTGAAGTATATGAGAAGAATGATTAAGGATCCCGTTTTTCTTACTTATA

TATTTATACCAATTGATTGTATTTATAACTGTAAAAATGTGTATGTTGTGTGCATATTTT

TTTTTGTGCATGCACATGCATGTAAATAGCTAAAATTATGAACATTTTATTTTTTGTTCA

GAAAAAAAAAACTTTACACACATAAAATGGCTAGTATGAATAGCCATATTTTATATAAAT

TAAATCCTATGAATTTATGACCATATTAAAAATTTAGATATTTATGGAACATAATATGTT

TGAAACAATAAGACAAAATTATTATTATTATTATTATTTTTACTGTTATAATTATGTTGT

CTCTTCAATGATTCATAAATAGTTGGACTTGATTTTTAAAATGTTTATAATATGATTAGC

ATAGTTAAATAAAAAAAGTTGAAAAATTAAAAAAAAACATATAAACACAAATGATGTTTT

TTCCTTCAATTTCGATGGGTACCCTCGAGGCTAGCGATATCTGATCACCCGGGGCGGCCG

CG

Restriction sites to linearize plasmid

Partial or complete disruption of the gene Partial

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite hdhfr

Promoter of the selectable marker eef1a

Selection (positive) procedure WR99210

Selection (negative) procedure No

Additional remarks genetic modification The construct to disrupt the ccp3 (pbsr, lap1) gene has been introduced into the genome of mutant 429cl1 (RMgm-121) that contains a disrupted ccp1/lap2 gene and the tgdhfr selectable marker.
The ccp3 gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the hdhfr selectable marker. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.

Additional remarks selection procedure Because the mutant contained the tgdhfr selectable marker in its genome (used to disrupt the ccp1/lap2 locus) the ccp3 gene has been disrupted using a construct that contains the hdhfr selectable marker that provides resistance against WR99210.

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 1	5′-AAGCTTATTTGTTCAGAAGGGTGGAC
Additional information primer 1
Sequence Primer 2	5′-CTGCAGTCCTTCACACAAACTATGAG
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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