RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5344
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1212600; Gene model (P.falciparum): PF3D7_1014200; Gene product: male gamete fusion factor HAP2, putative (HAP2; GCS1, Generative Cell Specific 1)
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, 18:08
  *RMgm-5344
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-5344
Principal nameΔhap
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 and gamete production is not affected. The phenotype analyses indicate a role of HAP2 in fertilisation. Female gametes are fertile, whereas male gametes are sterile.
Fertilization and ookineteMale and female gametocyte and gamete production is not affected. The phenotype analyses indicate a role of HAP2 in fertilisation. Female gametes are fertile, whereas male gametes are sterile.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of HAP2 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)
In Arabidopsis the male-specific sterility protein HAP2 has been identified by a genetic screen (HAP mutants: containing haploid-disrupting (hapless) mutations; Johnson M.A. et al., 2004, Genetics 168, 971-82) . A HAP2 family member called GCS1 (for generative cell-specific) was subsequently identified in a screen for lily genes whose transcripts were up-regulated in sperm (generative cells) . HAP2 is conserved and members were found in rice, Chlamydomonas, a red alga, a slime mold, Plasmodium falciparum, and Leishmania major.
Phenotype analyses of Plasmodium mutants lacking expression of HAP2/GCS1  (RMgm-155, RMgm-156) indicate a role of HAP/GCS1 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. Male gametes are able to attach to female gametes and form tight prefusion membrane attachments but the membranes of the gametes do not merge or fuse. As a result no zygotes/ookinetes are formed.

Phenotype
Male and female gametocyte and gamete production is not affected. The phenotype analyses indicate a role of HAP2 in fertilisation. Female gametes are fertile, whereas male gametes are sterile. 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_1212600
Gene Model P. falciparum ortholog PF3D7_1014200
Gene productmale gamete fusion factor HAP2, putative
Gene product: Alternative nameHAP2; GCS1, Generative Cell Specific 1
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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