RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4999
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1422900; Gene model (P.falciparum): PF3D7_0814600; Gene product: PIMMS22 protein, putative (PIMMS22)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 24 May 2021, 15:21
  *RMgm-4999
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) : 33791240
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line that expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherUkegbu CV, Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4999
Principal nameΔc22
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystOokinete motility comparable to wild type. No evidence for ookinete defects in invasion and traversal of the midgut epithelium. Strongly reduced oocyst numbers
SporozoiteStrongly reduced sporozoite numbers. No infection of mice by the bite of infected mosquitoes
Liver stageNo infection of mice by the bite of infected mosquitoes
Additional remarks phenotype

Mutant/mutation
The mutant Δc22 lacks expression of PIMMS22 and expresses GFP under control of the constitutive eef1a promoter

Protein (function)
PfPIMMS22 encodes a 393 amino acid (45 kDa) protein and PbPIMMS22 encodes a 393 aa (44 kDa) protein, both with no predicted signal peptide or transmembrane domain. The protein is highly conserved amongst Plasmodium orthologues. PyPIMMS22 (PY17X_1424900) was previously identified in salivary gland sporozoites through a  subtractive hybridization (SSH) profiling and termed sporozoite protein S15. The same protein was also identified in midgut oocyst sporozoites as an interacting partner to the apicomplexan specific RNA-binding protein, ALBA4, which is involved in mRNA regulation in gametocyte and midgut oocyst sporozoite development. PIMMS22 homologues are found in other apicomplexan parasites including Toxoplasma gondii, Neospora caninum and Eimeria with sequence identities to PIMMS22 ranging from 36% to 42%. InterPro domain analysis revealed no recognizable domain in PIMMS22.

Phenotype
Ookinete motility comparable to wild type. Evidence is presented for ookinete defects in invasion and traversal of the midgut epithelium (although this cannot completely explain the strongly reduced oocysts numbers, indicating reduced ookinete to oocyst transition after midgut traversal). Strongly reduced oocyst numbers. Strongly reduced sporozoite numbers. No infection of mice by the bite of infected mosquitoes.
(injection of mature ookinetes into the thorax could not rescue the sporozoite phenotype)

Additional information
Three genes were selected for analysis that exhibit highly abundant transcripts 24 h post blood feeding in the midguts of Anopheles coluzzii mosquitoes.
 
PIMMS01: PF3D7_0112100; PBANKA_0201700 
PIMMS57: PF3D7_1244500; PBANKA_1457700
PIMMS22: PF3D7_0814600; PBANKA_1422900 
((PIMMS: Plasmodium Infection of the Mosquito Midgut Screen)

Names of the corresponding gene-deletion mutants: Δc01, Δc57, Δc22

PbPIMMS01 and PbPIMMS57 transcripts were highly abundant in purified mature ookinetes and not detectable in mixed blood stages and purified gametocytes. While PbPIMMS57 appears to be specific for ookinetes, low levels of PbPIMMS01 transcripts are also detected in mature oocysts and midgut sporozoites at 10 days and 15 days post blood-feeding. Like PfPIMMS22, expression of PbPIMMS22 starts in gametocytes and continues at high levels at 24 hour and peaks in midgut sporozoites at day 15

From the abstract:
PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. 

Other mutants

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1422900
Gene Model P. falciparum ortholog PF3D7_0814600
Gene productPIMMS22 protein, putative
Gene product: Alternative namePIMMS22
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
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor partial disruption of Pbc01 in the c507 line, a 432 bp ApaI/HindIII 5’ homology arm and a 1102 bp EcoRI/BamHI 3’ homology arm was amplified using the primer pairs P24/P25 and P26/P27, respectively. For partial disruption of Pbc57 in the c507 line, a 590 bp ApaI/HindIII 5’ homology arm and a 684 bp EcoRI/BamHI 3’ homology arm was amplified using the primer pairs P28/P29 and P30/P31, respectively. For partial disruption of Pbc22 in the c507 line, a 560 bp ApaI/HindIII 5’ homology arm and a 712 bp EcoRI/BamHI 3’ homology arm was amplified using the primer pairs P32/P33 and P34/P35, respectively. These fragments were cloned into the pBS-TgDHFR vector. The targeting cassettes by ApaI/BamHI digestion allows knockout of 39% of Pbc01 CDS, 70% of Pbc57 CDS, and 80% of Pbc22 CDS.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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