RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4559
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0306000; Gene model (P.falciparum): PF3D7_0208900; Gene product: 6-cysteine protein (230p, P230p)
DisruptedGene model (rodent): PBANKA_0306100; Gene model (P.falciparum): PF3D7_0209000; Gene product: 6-cysteine protein | transmission-blocking target antigen s230 (P230)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 25 October 2018, 13:30
  *RMgm-4559
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30297725
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). The gfp-luciferase is integrated into the p230p gene locus.
The mutant parasite was generated by
Name PI/ResearcherMarin-Mogollon C, Janse CJ, Khan SM
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-4559
Principal name2764cl3
Alternative namePbΔp230Δp230p
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteThe double KO mutant PbΔp230Δp230p produces normal numbers of gametocytes and male gametocytes form male gamete numbers (by the process of exflagelllation) comparable to that of wild type parasites. In addition male gametes attach to red blood cells (forming exflagellation centers) but fail to recognise/attach to female gametes.
Fertilization and ookineteMale gametes are strongly affected in their fertility, resulting in nearly complete inhibition of ookinete formation in vitro (>0.99%); Motile males fail to attach to and penetrate female gametes.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
This mutant lacks expression of both p230 and p230p and expresses GFP-luciferase under the control of the constitutive eef1a promoter.
The p230 gene is disrupted in parasites of the the 676m1cl1 (RMgm-29) that contains a  GFP-luciferase expression cassette integrated into the p230p gene. This  line does not contain a drug-selectable marker. The p230 gene is disrupted using a construct that was used to generate mutant RMgm-350.

Phenotype
In rodent malaria parasites both P230 and P230p are expressed in male gametocytes/gametes.
The single p230p KO mutant produces fertile gametes, that attach to red blood cells (forming exflagellation centers) and shows a phenotype throughout the complete life cycle that is not different from that of wild type parasites (see also PMID 20386715).
The single p230 KO mutant produces gametes that are NOT fertile. They attach to red blood cells (forming exflagellation centers) but fail to recognise/attach to female gametes (see also PMID 20386715 and RMgm-350).

The double KO mutant PbΔp230Δp230p produces normal numbers of gametocytes and male gametocytes form male gamete numbers (by the process of exflagelllation) comparable to that of wild type parasites. In addition, male gametes attach to red blood cells (forming exflagellation centers) but fail to recognise/attach to female gametes.

Additional information
From the paper:
'The role of P230 and P230p in P. falciparum gamete binding to RBC is different to the role of these proteins in the rodent parasite P. berghei (see PMID 30297725) . Single gene-deletion mutants lacking expression of either P230 or P230p in P. berghei exhibit formation of exflagellation centres like WT, indicating that male gametes of these mutants bind normally to RBC. To examine a possible compensatory role in RBC binding of the two P. berghei paralogs we generated a p230 and p230p double gene deletion mutant. Activated male gametocytes of this mutant, PbΔp230Δp230p, formed WT-like levels of exflagellation centres demonstrating an absence of a role of these proteins in RBC binding of P. berghei male gametes'.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0306000
Gene Model P. falciparum ortholog PF3D7_0208900
Gene product6-cysteine protein
Gene product: Alternative name230p, P230p
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
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

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0306100
Gene Model P. falciparum ortholog PF3D7_0209000
Gene product6-cysteine protein | transmission-blocking target antigen s230
Gene product: Alternative nameP230
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 genePartial
Additional remarks partial/complete disruption Of the 4486bp coding region, nucleotides 831-1410 were disrupted
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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