Back to search resultsSummaryRMgm-4068
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Successful modification | The 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 | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Kublin JG, Kappe SH |
Name Group/Department | Center for Infectious Disease Research |
Name Institute | Center for Infectious Disease Research |
City | Seattle |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-4068 |
Principal name | Py p52−/p36−/sap1− |
Alternative name | PyGAP3KO |
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 | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Complete (early) arrest of growth of liver stages within hepatocytes |
Additional remarks phenotype | Mutant/mutation Additional information |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1003600 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0404500 | ||||||||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||||||||
Gene product: Alternative name | P36p; P52 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||
Additional remarks genetic modification | A 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
Primer information: Primers used for amplification of the target sequences
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1003500 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0404400 | ||||||||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||||||||
Gene product: Alternative name | P36 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||
Additional remarks genetic modification | A 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
Primer information: Primers used for amplification of the target sequences
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_0903500 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1147000 | ||||||||||||||||||||||||
Gene product | sporozoite and liver stage asparagine-rich protein | sporozoite asparagine-rich protein | ||||||||||||||||||||||||
Gene product: Alternative name | SAP1, SLARP; sporozoite (and liver stage) asparagine-rich protein; S22 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||
Additional remarks genetic modification | A 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
Primer information: Primers used for amplification of the target sequences
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