RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5220
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PbANKA_1343400; Gene model (P.falciparum): PF3D7_1328200; Gene product: conserved Plasmodium protein, unknown function (BRCA2; breast cancer susceptibility protein 2)
Transgene
Transgene not Plasmodium: GFP
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)
Insertion locus: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 13 July 2022, 13:58
  *RMgm-5220
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) : 35804459
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherYoshikawa Y, Ikadai H
Name Group/DepartmentLaboratory of Veterinary Parasitology, School of Veterinary Medicine
Name InstituteKitasato University
CityTowada, Aomori
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5220
Principal namePbBrca2‑KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stage(Slightly) reduced growth/multiplication of asexual blood-stage parasites in mice.
Gametocyte/Gamete(Slightly) reduced formation of female gametocytes
Fertilization and ookineteReduced ookinete production. Mature ookinetes formed had a similar (light-microscope) morphology as wild-type ookinetes.
OocystReduced oocyst production. Presence of degenerated, vacuolated oocysts. No sporozoite formation inside oocysts.
SporozoiteNo salivary gland sporozoites
Liver stageNo infection of mice by mosquito bite.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Brca2 and expresses GFP under the constitutive hsp70 promoter

Protein (function)
Homologous recombination (HR) occurs during meiosis in Plasmodium spp., which involves the DNA recombinases DNA repair protein Rad51 homolog 1 (Rad51) and DNA meiotic recombinase 1 (Dmc1). It was reported that Dmc1 knockout (KO) in Plasmodium berghei resulted in a reduced number and altered development of oocysts and sporozoites. In humans, Rad51 and Dmc1 are required for HR and are regulated by breast cancer susceptibility protein 2 (BRCA2). Most eukaryotes harbor BRCA2 homologs, such as mammals, Xenopus, fish, Caenorhabditis elegans, Ustilago maydis, Trypanosoma, and Leishmania. They are absent in yeasts, archaea, and bacteria. In lower eukaryotes such as C. elegans, U. maydis, Trypanosoma, and Leishmania, Brca2 orthologs possess only the vital BRC repeats and the tower and oligonucleotide/oligosaccharide-binding (OB)-fold domains. 
In this study It was predicted that Brca2 in P. berghei (PBANKA_1343400) has three BRC repeats and OB-fold and tower domains such as Trypanosoma brucei and Leishmania infantum Brca2 and Ustilago maydis Brh2, which are the simplest BRCA2 orthologs harboring essential BRCA2 domains, i.e. one BRC repeat, OB and tower domains, and nuclear localization signals. 

Phenotype
(Slightly) reduced growth/multiplication of asexual blood-stage parasites in mice.
(Slightly) reduced formation of female gametocytes. Reduced ookinete production. Mature ookinetes formed had a similar (light-microscope) morphology as wild-type ookinetes.
Reduced oocyst production. Presence of degenerated, vacuolated oocysts. No sporozoite formation inside oocysts. No salivary gland sporozoites. No infection of mice by mosquito bite.

Additional information
Evidence is presented that: 
- PbBrca2 BRC repeats interacted only with PbRad51 

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_1343400
Gene Model P. falciparum ortholog PF3D7_1328200
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameBRCA2; breast cancer susceptibility protein 2
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 parasitepuromycin-N-acetyltransferase (pac)
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSee mutant RMgm-4180 for the transfection and selection method (selection with pac).

PbBrca2 was knocked out by double-crossover HR technology using a pBluescript II-based plasmid containing a puromycin resistance gene inserted upstream (5′) of the PbEf1α gene and downstream (3′) of the PbDhfr-ts gene. A donor plasmid with 1000-bp homology arms was cloned upstream with the primers 5′-PbBrca2 forward (F) (5′-CGGGGTACCTTTTATTGTATCCCTATAA-3′), and 5′-PbBrca2 reverse (R) (5′-GGCGGGCCCCTTTATTTAATTATTAAGATTTTTTTGTTAC-3′); and cloned downstream with the primers 3′ PbBrca2 F (5′-CCGTCTAGATATCGTTTTAAGGTTGTTTC-3′), and 3′-PbBrca2 R (5′-CCGGCGGCCGCTATGTTGTATTGTTTGTTTT-3′). To clone the homology arms, the plasmids were treated with the restriction enzymes KpnI and ApaI for upstream of the PbBrca2 gene and XbaI and NotI for downstream of the PbBrca2 gene . Ten micrograms of donor vector was linearized using ScaI and used for transfection
Additional remarks selection procedureSee mutant RMgm-4180 for the transfection and selection method (selection with pac).
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant DHFR-TS gene. A transgenic GFP-expressing parasite clone (named WT:GFP) was obtained by inserting this construct downstream of the dhfr/ts locus by homologous recombination.
Additional remarks selection procedure
Other details transgene
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
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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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