RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-816
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1321700; Gene model (P.falciparum): PF3D7_1458000; Gene product: cysteine proteinase falcipain 1 (FP1; berghepain 1 (BP1))
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1
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 Asexual bloodstage;
Last modified: 23 December 2016, 17:44
  *RMgm-816
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) : 25941254
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 1037m1f1mocl1 (RMgm-32)
Other information parent lineP. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 20019192).
The mutant parasite was generated by
Name PI/ResearcherJ. Lin; C.J. Janse; S.M. Khan
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-816
Principal name2249cl1; 2250cl1
Alternative name∆bp1
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageReduced growth rate of blood stages (normal production of hemozoin)
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of berghepain 1 (BP1)

Protein (function)
P. falciparum expresses four cysteine proteases namely, falcipain-1 (FP-1), falcipain-2 and -2' (FP-2A and FP-2B) and falcipain-3 (FP-3) at asexual blood stages of the parasite. These proteases perform multiple functions such as hemoglobin hydrolysis, erythrocyte rupture and erythrocyte invasion and differ in their timing of expression. Falcipain-1 is active at the invasive merozoite stage while falcipain-2/-2' and -3 are expressed mainly at early and late trophozoite stages respectively.

Both P. falciparum and P. berghei have an papain-like cysteine protease encoded by a syntenic gene (falcipain 1, FP1, PF3D7_1458000 and berghepain 1, BP1, PBANKA_132170). Evidence has been presented that this protease is located (and active) in the (apical end) of merozoites and not in the digestive vacuole, suggesting that it has no role in hemoglobin digestion.

P. berghe has an additional papain-like cysteine protease, Berghepain-2 (BP2), which is equivalent to the three P. falciparum digestive vacuole falcipains (papain-like cysteine endoproteases), FP-2 (FP2a, PF3D7_1115700; FP2b, PF3D7_1115300) and -3 (PF3D7_1115400). The gene encoding BP2 has a syntenic location to the three falcipain genes of P. falciparum and also the the single copy vivapain-2 of P. Vivax (PVX_086040).

Phenotype
Blood stages showed reduced growth/multiplication in mice (but produced normal levels of hemozoin)

Additional information



Figure: Generation of the P. berghei mutants ∆pm4 (RMgm-808), ∆bp1 (RMgm-816) and ∆bp2 (RMgm-809).
(primer sequences can either be found in Lin et al. (2015). J. Exp. Med. or obtained from the Leiden Malaria Research Group).
(A) Schematic representation of gene-deletion constructs targeting the open reading frame (ORF) of genes expressing plasmepsin 4 (pm4), berghepain 2 (bp2) or berghepain 1 (bp1) by double cross-over homologous recombination, and wild-type (wt) gene loci before and after disruption. The constructs contain a drug-selectable marker cassette (SM; black box) and gene target regions (hatched boxes). Primer positions (arrows) for diagnostic PCRs are shown (see Table S4 for primer sequences and expected product sizes). (B) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm correct disruption of pm4 in mutant ∆pm4-b. Northern analysis of blood-stage mRNA (right) confirms the absence of pm4 transcripts in the ∆pm4-b mutant. The following primers were used for diagnostic PCRs: 5' integration (5’ in): L5516/L4096; 3' integration: (3’ in) L1662/L5517; SM (hdfhr::yfcu): L4698/L4699; pm4 ORF: L5518/L5519. Separated chromosomes were hybridized using an hdhfr probe that recognizes the DNA-construct integrated into the pm4 locus on chromosome 10. Northern blot was hybridized using a PCR probe recognizing the pm4 ORF (primers L5518/L5519) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (C) Diagnostic PCR (left) confirms the correct deletion of the bp2 gene in mutant ∆bp2-a. RT-PCR analysis of blood stage mRNA (right) shows the absence of bp2 transcription in ∆bp2-a blood-stages. The following primer pairs were used for diagnostic PCR analyses: 5’ in, RS835/RS32; 3’ in, RS110/RS836; SM (tgdhfr/ts), RS404/RS405; bp2 ORF, RS514/RS515. For RT-PCR the following primers were used: tub (tubulin), RS782/RS783 and bp2, RS515/RS516. (D) Diagnostic PCR (left) and Southern analysis of pulsed field gel-separated chromosomes (center) confirm the correct disruption of the bp2 gene in mutant ∆bp2-b. Northern blot analysis of blood stage mRNA (right) confirms the absence of bp2 transcripts in ∆bp2-b. The following primers were used for diagnostic PCRs: 5’ in, L5024/L3211; 3’ in, L5025/L1662; SM (hdhfr::yfcu), L4698/L4699; bp2 ORF, L5026/L5027. Separated chromosomes were hybridized using an hdhfr probe that recognizes the DNA-construct integrated into the bp2 locus on chromosome 9. Northern blot was hybridized using a PCR probe recognizing the bp2 ORF (primers L5026/L5027) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). (E) Southern analysis of pulsed field gel-separated chromosomes (left) confirms the correct disruption of bp1 in mutant ∆bp1. Northern analysis of blood-stage mRNA (right) confirms the absence of bp1 transcripts in mutant ∆bp1. Separated chromosomes were hybridized using an 3’UTR pbdhfr probe that recognizes the DNA-construct integrated into the bp1 locus on chromosome 13, the endogenous dhfr/ts on chromosome 7 and the GFP-luciferase reporter cassette in the 230p locus on chromosome 3. Northern blot was hybridized using a PCR probe recognizing the bp1 ORF (primers L7422/L7423) and with an oligonucleotide probe L644R recognizing the large subunit rRNA (as loading control). See Table S4 for primers used for generation of probes.

Other mutants
RMgm-809: A mutant lacking expression of berghepain 2 (BP2)
RMgm-817: A double ko mutant lacking expression of PM4 (Plasmepsin 4) and berghepain 2 (BP2)


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1321700
Gene Model P. falciparum ortholog PF3D7_1458000
Gene productcysteine proteinase falcipain 1
Gene product: Alternative nameFP1; berghepain 1 (BP1)
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct used to disrupt the bp1 locus was obtained from Photini Sinnis.
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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 KspI (SacII)
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerama-1
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase 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_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative name
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