RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5277
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1030100; Gene model (P.falciparum): PF3D7_1412500; Gene product: actin II (actin2)
Transgene
 Transgene not Plasmodium: mCherry-3xMyc
Promoter: Gene model: PBANKA_1030100; Gene model (P.falciparum): PF3D7_1412500; Gene product: actin II (actin2)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1030100; Gene product: actin II (actin2)
Phenotype Gametocyte/Gamete;
Last modified: 9 January 2023, 16:47
  *RMgm-5277
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) : 22705315
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 Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherKooij, T.W.A.; Rauch, M.M.; Matuschewski, K.
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-5277
Principal nameact2−::mCherry
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/GameteGametocyte conversion rates, male:female ratios, and exflagellation rates of act2−::mCherry were comparable to those of wild type parasites. In contrast, male exflagellation of act2−::mCherry was completely abrogated, reproducing the act2−phenotype.
Ring and trophozoite stage parasites showed virtually undetectable levels of mCherry expression. mCherry expression is considerably higher in male gametocytes compared to female gametocytes.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Actin II and expresses mCherry-3xmyc under control of the actin II promoter. It does not contain a drug-selectable marker that has been removed by negative selection.

Protein (function)
Actin, a cytoskeletal protein, has many diverse functions in eukaryotic cells ranging from roles in cell motility, cell division, vesicle trafficking to functions in cell signaling and regulation of transcription. Plasmodium parasites express two actins, Actin I and Actin II. Actin II is mainly transcribed/expressed in malae gametocytes/gametes

Phenotype
Gametocyte conversion rates, male:female ratios, and exflagellation rates of act2−::mCherry were comparable to those of wild type parasites. In contrast, male exflagellation of act2−::mCherry was completely abrogated, reproducing the act2−phenotype.
Ring and trophozoite stage parasites showed virtually undetectable levels of mCherry expression. mCherry expression is considerably higher in male gametocytes compared to female gametocytes.

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_1030100
Gene Model P. falciparum ortholog PF3D7_1412500
Gene productactin II
Gene product: Alternative nameactin2
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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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationWe also designed and created a new construct targeting PbACT2 using the pBAT-SIL6 vector. Two targeting sequences homologous to the 5′ and 3′UTR flanking regions of PbACT2 were cloned into the multiple cloning sites, and the vector was linearized by SalI restriction digestion. Following successful integration through ends-out recombination, the PbACT2 promoter region was directly linked to the mCherry-3xMyc sequence. Hence, the vector effectively functioned as a gene deletion construct and a promoter activity reporter.
We transfected the clonal, recycled non-fluorescent 6rec parasites, because this would allow us to verify the functioning of the GFP cassette in this approach. The vector integrated readily and we were able to isolate the recombinant parasites. Purity of the final act2−::mCherry population was verified by PCR and Southern analysis.
Following recycling of the drug-selectable cassette via negative selection and subsequent cloning by limiting dilution, 6rec parasites have restored their pyrimethamine-sensitivity rendering them amendable to a new round of genetic manipulation.
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-3xMyc
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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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationWe also designed and created a new construct targeting PbACT2 using the pBAT-SIL6 vector. Two targeting sequences homologous to the 5′ and 3′UTR flanking regions of PbACT2 were cloned into the multiple cloning sites, and the vector was linearized by SalI restriction digestion. Following successful integration through ends-out recombination, the PbACT2 promoter region was directly linked to the mCherry-3xMyc sequence. Hence, the vector effectively functioned as a gene deletion construct and a promoter activity reporter.
We transfected the clonal, recycled non-fluorescent 6rec parasites, because this would allow us to verify the functioning of the GFP cassette in this approach. The vector integrated readily and we were able to isolate the recombinant parasites. Purity of the final act2−::mCherry population was verified by PCR and Southern analysis.
Following recycling of the drug-selectable cassette via negative selection and subsequent cloning by limiting dilution, 6rec parasites have restored their pyrimethamine-sensitivity rendering them amendable to a new round of genetic manipulation.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1030100
Gene Model P. falciparum ortholog PF3D7_1412500
Gene productactin II
Gene product: Alternative nameactin2
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 Not available
Gene productNot available
Gene product: Alternative name
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_1030100
Gene productactin II
Gene product: Alternative nameactin2
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