RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4068
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1003600; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; P52)
DisruptedGene model (rodent): PY17X_1003500; Gene model (P.falciparum): PF3D7_0404400; Gene product: 6-cysteine protein (P36)
DisruptedGene model (rodent): PY17X_0903500; Gene model (P.falciparum): PF3D7_1147000; Gene product: sporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein (SAP1, SLARP; sporozoite (and liver stage) asparagine-rich protein; S22)
Phenotype Liver stage;
Last modified: 10 January 2017, 19:13
  *RMgm-4068
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28053159
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherKublin JG, Kappe SH
Name Group/DepartmentCenter for Infectious Disease Research
Name InstituteCenter for Infectious Disease Research
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4068
Principal namePy p52−/p36−/sap1−
Alternative namePyGAP3KO
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 stageComplete (early) arrest of growth of liver stages within hepatocytes
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of three proteins: P52, P36 and SAP1

Protein (function)

Phenotype
Complete (early) arrest of growth of liver stages within hepatocytes.

Additional information

Other mutants
See PY17X_1003600  for other P52 mutants
See PY17X_1003500  for other P36 mutants
See PY17X_0903500  for other SAP1 mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1003600
Gene Model P. falciparum ortholog PF3D7_0404500
Gene product6-cysteine protein
Gene product: Alternative nameP36p; P52
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
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationA P. yoelii XNL parasite was created with deletions in p36, p52 and sap1. The gene-deletion approach was based on the published Gene Insertion/Marker Out (GIMO) strategy. Briefly, tandemly-arranged p36 and p52 genes were deleted from P. yoelii XNL using double crossover homologous recombination. The complementary regions of interest were ligated into pL0034, flanking the positive/negative selectable marker, to create plasmid pL0034_2KO. pL0034_2KO was linearized and after transfection and positive selection with pyrimethamine, parasites were cloned and gene knockouts confirmed using standard PCR methods. Deletion of p36 and p52 constituted the P. yoelii GAP2KO (Py GAP2KO) parasite. To create Py GAP3KO, the selectable marker was removed from pL0034_2KO and the plasmid was then recircularized, linearized and transfected into Py GAP2KO parasite. Negative selection with 5-flurocytosine selected for a population of parasites in which the selectable marker was removed. Parasites were cloned and removal of the selectable marker was confirmed by PCR, resulting in a markerless Py GAP2KO parasite (Py GAP2KO-m), which was amenable to further genetic manipulation. Using the same strategy for the initial gene deletions, complementary regions upstream and downstream of the sap1 gene were ligated between the selectable marker of plasmid pL0034 to create pL0034_sap1. Py GAP2KO-m was transfected with pL0034_sap1 under pyrimethamine selection. Knockout of sap1 was confirmed by PCR and subsequent parasite cloning isolated Py GAP3KO parasites for downstream phenotypic analysis. Two individual clones of Py GAP2KO-m parasites were analyzed throughout the life cycle and behaved as wild type until liver stage development.
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
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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1003500
Gene Model P. falciparum ortholog PF3D7_0404400
Gene product6-cysteine protein
Gene product: Alternative nameP36
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
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationA P. yoelii XNL parasite was created with deletions in p36, p52 and sap1. The gene-deletion approach was based on the published Gene Insertion/Marker Out (GIMO) strategy. Briefly, tandemly-arranged p36 and p52 genes were deleted from P. yoelii XNL using double crossover homologous recombination. The complementary regions of interest were ligated into pL0034, flanking the positive/negative selectable marker, to create plasmid pL0034_2KO. pL0034_2KO was linearized and after transfection and positive selection with pyrimethamine, parasites were cloned and gene knockouts confirmed using standard PCR methods. Deletion of p36 and p52 constituted the P. yoelii GAP2KO (Py GAP2KO) parasite. To create Py GAP3KO, the selectable marker was removed from pL0034_2KO and the plasmid was then recircularized, linearized and transfected into Py GAP2KO parasite. Negative selection with 5-flurocytosine selected for a population of parasites in which the selectable marker was removed. Parasites were cloned and removal of the selectable marker was confirmed by PCR, resulting in a markerless Py GAP2KO parasite (Py GAP2KO-m), which was amenable to further genetic manipulation. Using the same strategy for the initial gene deletions, complementary regions upstream and downstream of the sap1 gene were ligated between the selectable marker of plasmid pL0034 to create pL0034_sap1. Py GAP2KO-m was transfected with pL0034_sap1 under pyrimethamine selection. Knockout of sap1 was confirmed by PCR and subsequent parasite cloning isolated Py GAP3KO parasites for downstream phenotypic analysis. Two individual clones of Py GAP2KO-m parasites were analyzed throughout the life cycle and behaved as wild type until liver stage development.
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
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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0903500
Gene Model P. falciparum ortholog PF3D7_1147000
Gene productsporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein
Gene product: Alternative nameSAP1, SLARP; sporozoite (and liver stage) asparagine-rich protein; S22
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
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationA P. yoelii XNL parasite was created with deletions in p36, p52 and sap1. The gene-deletion approach was based on the published Gene Insertion/Marker Out (GIMO) strategy. Briefly, tandemly-arranged p36 and p52 genes were deleted from P. yoelii XNL using double crossover homologous recombination. The complementary regions of interest were ligated into pL0034, flanking the positive/negative selectable marker, to create plasmid pL0034_2KO. pL0034_2KO was linearized and after transfection and positive selection with pyrimethamine, parasites were cloned and gene knockouts confirmed using standard PCR methods. Deletion of p36 and p52 constituted the P. yoelii GAP2KO (Py GAP2KO) parasite. To create Py GAP3KO, the selectable marker was removed from pL0034_2KO and the plasmid was then recircularized, linearized and transfected into Py GAP2KO parasite. Negative selection with 5-flurocytosine selected for a population of parasites in which the selectable marker was removed. Parasites were cloned and removal of the selectable marker was confirmed by PCR, resulting in a markerless Py GAP2KO parasite (Py GAP2KO-m), which was amenable to further genetic manipulation. Using the same strategy for the initial gene deletions, complementary regions upstream and downstream of the sap1 gene were ligated between the selectable marker of plasmid pL0034 to create pL0034_sap1. Py GAP2KO-m was transfected with pL0034_sap1 under pyrimethamine selection. Knockout of sap1 was confirmed by PCR and subsequent parasite cloning isolated Py GAP3KO parasites for downstream phenotypic analysis. Two individual clones of Py GAP2KO-m parasites were analyzed throughout the life cycle and behaved as wild type until liver stage development.
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
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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6