RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5514
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0920100; Gene model (P.falciparum): PF3D7_1128100; Gene product: prefoldin subunit 5, putative (PbPCS5)
Transgene
Transgene not Plasmodium: mcherry
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)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: Luciferase
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 (P230p; 230p)
Phenotype Oocyst; Sporozoite;
Last modified: 16 June 2024, 18:12
  *RMgm-5514
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38009402
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1320
Other information parent lineThis transgenic reporter line (1868cl1; RMgm-1320) expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Both reporter cassettes are introduced into the silent 230p locus, using a single DNA construct. This transgenic line does not contain a drug-selectable marker
The mutant parasite was generated by
Name PI/ResearcherBeyeler R, Heussler V
Name Group/DepartmentInstitute of Cell Biology
Name InstituteUniversity of Bern
CityBern
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-5514
Principal namePbPCS5-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystSlightly fewer oocysts compared to wild type. No decrease in oocyst numbers between day 14 and 21, indicating a defect in oocyst rupture and sporozoite egress. Oocyst size at day 7 and 10 smaller compared to wild type (and larger at day 14 and 21 compared to wild type). Inside oocysts PbPCS5-KO parasites form thinner and elongated sporozoites with fewer microtubules
SporozoiteNo midgut and salivary gland sporozoites could be detected
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of prefoldin subunit 5, putative and expresses mCherry and luciferase under control of constitutive promoters

Protein (function)
Prefoldin is a heterohexameric chaperone complex known to be involved in the assembly and maintenance of actin as well as α- and β-tubulins in a wide range of eukaryotes. P. berghei possesses 6 prefoldin subunits and since the function of prefoldin is conserved among many eukaryotic species, it may also be involved in the cytoskeletal assembly in Plasmodium, which is essential for sporozoite formation. Here, we investigated the function of subunit 5 of P. berghei prefoldin complex during Plasmodium mosquito stage development

Phenotype
Slightly fewer oocysts compared to wild type. No decrease in oocyst numbers between day 14 and 21, indicating a defect in oocyst rupture and sporozoite egress. Oocyst size at day 7 and 10 smaller compared to wild type (and larger at day 14 and 21 compared to wild type). Inside oocysts PbPCS5-KO parasites form thinner and elongated sporozoites with fewer microtubules. No midgut and salivary gland sporozoites could be detected.

Additional information
Evidence is presented that:
- PbPCS5 is dispensable for DNA replication but seems to be important at a later stage of sporogony.
- The microtubular organization is disturbed in PbPCS5-KO oocysts
- PbPCS5-KO parasites form thinner and elongated sporozoites with fewer microtubules
- PbPCS5-KO microtubules extend into the sporoblast during sporogony
- Subpellicular microtubules contact nuclei within sporoblast
From the paper: ' PbPCS5 has at least two functions during parasite development in mosquitoes: i) it is needed for the correct polymerization of subpellicular microtubules. In the absence of PbPCS5, fewer subpellicular microtubules are assembled that then reach into the sporoblast where they can contact and deform nuclei, and ii), PbPCS5 appears also to be required for achieving the correct organization of nuclei. Nuclei are seemingly unable to enter the forming sporozoites resulting in many anucleated sporozoites. This might be due to an effect on nucleus orientation or rootlet fibre assembly. Alternatively, the forming sporozoite buds might be too narrow for nuclei to be pulled in. PbPCS5-deficient sporozoites were unable to egress from oocysts and only mechanical disruption could release them from oocysts'.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0920100
Gene Model P. falciparum ortholog PF3D7_1128100
Gene productprefoldin subunit 5, putative
Gene product: Alternative namePbPCS5
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-342244
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/yfcu
Promoter of the selectable markereef1a
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemcherry
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-342244
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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 / 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct 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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
Additional remarks selection procedure
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 nameP230p; 230p
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