RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-349
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1359700; Gene model (P.falciparum): PF3D7_1346800; Gene product: 6-cysteine protein (P47; Pfs47)
DisruptedGene model (rodent): PBANKA_1359600; Gene model (P.falciparum): PF3D7_1346700; Gene product: 6-cysteine protein | transmission blocking target antigen precursor (P48/45)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 11 August 2010, 22:20
  *RMgm-349
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) : 20386715
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherM.R. van Dijk; C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-349
Principal name192cl1; 203 (uncloned)
Alternative nameΔp47-Δ48/45
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal numbers of male and female gametocytes are produced. Male and female gamete formation is normal (escape from host red blood cell, formation of 8 motile male gametes). Female and male 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.
Fertilization and ookineteFemale and male 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.
Mutant parasites produce low numbers of ookinetes in vivo (Anopheles stephensi). See also 'Additional remarks phenotype'
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of both P47 and P48/45.

Protein (function)
The P47 and P48/45 proteins are members of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins. The proteins are characterized by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organization throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites (see also 'Additional Information'). P47 is specifically expressed on the surface of female gametocytes/gametes (no expression in male gametocytes/gametes) and P48/45 is expressed on the surface of both male and female gametocytes/gametes.

Phenotype
Phenotype analyses demonstrate that both P48/45 (male fertility factor) and  P47 (female fertility factor) play an important role in fertilization. See also mutants RMgm-15 and RMgm-348 that lack either P48/45 or P47. Motile males fail to attach to and penetrate female gametes (see phenotype description).

Additional information
The p47 gene forms a paralogous gene pair with the gene p48/45  which are closely linked in the genome. In this mutant the genes encoding P48/45 and P47 have been disrupted (double knock-out mutant) using a single DNA construct.

The mutant  shows very low levels of fertilization with higher fertilization rates in the mosquito than in in vitro ookinete cultures. Mutant parasites produce low numbers of ookinetes and oocysts in Anopheles stephensi mosquitoes (see also the phenotype description 'Fertilization and ookinete'). Both the ookinetes and oocysts show a normal morphology and oocysts produce normal numbers of sporozoites that are infectious to mice.

Table: P. falciparum gene members of the 6-cys family

Gene P. falciparum Gene P. falciparum
p48/45  PF13_0247 p12p  PFF0620c
p47  PF13_0248 p230p  PFB0400w
p36  PFD0210c p230  PFB0405w
p52  PFD0215c p38  PFE0395c
p12  PFF0615c p41  PFD0240c

Other mutants
RMgm-15: A mutant lacking expression of P48/45
RMgm-345: The mutant lacks expression of the P48/45 protein and expresses GFP under the control of a male specific promoter and RFP under the control of a female specific promoter. This mutant has been generated to be able to distinguish the female gametocytes/gametes and ookinetes based on their RFP expression in cross-fertilisation experiments with fertile males of other mutants and for determination of meiosis in cross-fertilisation studies. Meiosis in zygotes is determined by FACS analysis of the DNA content of Hoechst-stained gametes/zygotes selected based on their RFP-fluorescence.
RMgm-346: An independent mutant lacking expression of P48/45 which expresses GFP under the constitutive eef1a promoter. This mutant shows the same phenotype as other mutants lacking expression of P48/45. This mutant has been generated to be able to distinguish the female gametocytes and ookinetes based on their high GFP expression in cross-fertilisation experiments with fertile males of other mutants.
RMgm-347: A mutant lacking expression of P47 that expresses GFP under the constitutive eef1a promoter
RMgm-348: A mutant lacking expression of P47


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1359700
Gene Model P. falciparum ortholog PF3D7_1346800
Gene product6-cysteine protein
Gene product: Alternative nameP47; Pfs47
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 The 5'UTR and 260bp of the coding region remain intact. The same 5'target region was used as with the p47 single ko (see RMgm-348)
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn this mutant the genes encoding P48/45 and P47 have been disrupted (double knock-out mutant) using a single DNA construct.
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_1359600
Gene Model P. falciparum ortholog PF3D7_1346700
Gene product6-cysteine protein | transmission blocking target antigen precursor
Gene product: Alternative nameP48/45
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn this mutant the genes encoding P48/45 and P47 have been disrupted (double knock-out mutant) using a single DNA construct.
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