RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-5343
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0933700; Gene model (P.falciparum): PF3D7_1113900; Gene product: mitogen-activated protein kinase 2 (MAP2; MAP-2; MAPK2)
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 15 September 2023, 18:07
  *RMgm-5343
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) : 37708854
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-928
Other information parent lineThe mutant line 1804cl1 (RMgm-828) expresses mCherry under the control of the P. berghei hsp70 promoter. The mutant does not contain a drug-selectable marker.
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The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D, Christophides GK
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-5343
Principal nameΔmap2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteMale and female gametocyte production is not affected. No male gamete formation (absence of exflagellation). Female gametes NOT affected and fertile
Fertilization and ookineteNo male gamete production. No fertilization.
Female gametes NOT affected and fertile (as shown by cross-fertilization studies).
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MAP2 and expresses the reporter mCherry under control of the constitutive hsp70 promoter. It does not contain a drug-selectable marker that has been removed by negative selection.

Protein (function)
MAP kinases are serine/threonine protein kinases involved in a variety of functions including cell proliferation. In Plasmodium two MAPK proteins have been identified, MAPK1 (MAP1) and MAPK2 (MAP2).
Using reverse genetics approaches MAPK1 was shown to be dispensable during blood and mosquito stage development in both P. falciparum and P. berghei  (RMgm-526). While MAPK2 was found to be dispensable in asexual blood stages but essential for male gametogenesis in P. berghei (RMgm-62,RMgm- 63, RMgm-66), a vital function of this kinase in  asexual blood stage parasites was demonstrated in P. falciparum. Absence of the mitogen-activated protein kinase MAP2 inhibits male gamete exflagellation by impairing cytokinesis and axoneme motility 

Phenotype
Male and female gametocyte production is not affected. No male gamete formation (absence of exflagellation). No fertilization. Female gametes NOT affected and fertile (as shown by cross-fertilization studies).

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0933700
Gene Model P. falciparum ortholog PF3D7_1113900
Gene productmitogen-activated protein kinase 2
Gene product: Alternative nameMAP2; MAP-2; MAPK2
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
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 modificationFor the generation of the Δmap2 and Δhap2 background lines, we used the PlasmoGEM vectors PbGEM-111778 and PbGEM-102303, respectively. A total of 5 μg of each plasmid was used to transfect segmented P. berghei schizonts as previously described. Transgenic parasites were selected with 0.07 mg/mL pyrimethamine (Sigma) in drinking water from day 1 pi. Disruption was confirmed in the resistant parasite populations by PCR and clonal lines were derived by limiting dilution. To allow the use of Δmap2 and Δhap2 as background lines in the screen, we induced excision of the resistance cassette from the genome using negative selection, through the administration of 5 fluorocytosine (1 mg/mL, Sigma) via the drinking water.
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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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
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThis reporter mutant expressing mCherry does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
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3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren