RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4621
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1312600; Gene model (P.falciparum): PF3D7_1448900; Gene product: conserved protein, unknown function
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 (230p)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 20 November 2019, 15:23
  *RMgm-4621
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) : 31730853
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 expresses luciferase under the control of the eef1α (PBANKA_113330) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_071190) promoter. Both reporter cassetes 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/ResearcherStanway RR, Janse CJ, Heussler VT (1)
Name Group/DepartmentInstitute of Cell Biology
Name InstituteUniversity of Bern
CityBern
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4621
Principal name2844cl1
Alternative nameΔPBANKA_1312600
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 ookineteAberrant ookinete formation: many 'comma-shaped' zygotes/ookinetes; many zygotes with only small protrusions; pigment often not clustered in crystalloid bodies, but dispersed; many zygotes with large (4N?) nuclei; but also with smaller (2N?) nuclei.
OocystNo formation of oocysts
SporozoiteNo formation of oocysts; no formation of salivary gland sporozoites
Liver stageSee Additional Information: Evidence for a role during liver stage development
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PBANKA_1312600 and expresses mCherry and luciferase under constitutive promoters

Protein (function)
The gene has been selected based on a predicted role in mosquito- or liver-stage development in a high throughput screen where more than 1300 barcoded P. berghei mutants were followed through the complete life cycle.

Phenotype
Aberrant ookinete formation: many 'comma-shaped' zygotes/ookinetes; many zygotes with only small protrusions; pigment often not clustered in crystalloid bodies, but dispersed; many zygotes with large (4N?) nuclei; but also with smaller (2N?) nuclei.
No formation of oocysts; no formation of salivary gland sporozoites

Additional information
The gene has been selected based on a predicted role in mosquito- or liver-stage development in a high throughput screen where more than 1300 barcoded P. berghei mutants (PlasmoGem) were followed through the complete life cycle.
Barcodes were quantified in blood stages (B1), midgut oocysts (MG), salivary gland sporozoites (SG) and in blood stages after intravenous infection of mice with sporozoites (B2).
In the different life cycle transitions, barcode quantification was described as not-reduced, reduced or no power. For this gene:
B1-MG: reduced
MG-SG: no power
SG-B2: reduced (-10.2)

Cross-fertilization between different mutants in the mosquito midgut limits the power of the screen to reveal gene functions during the subsequent diploid and polyploid stages (i.e. zygotes, ookinetes and oocysts). For instance, knock-out mutants in which only one sex is sterile, can transmit their barcodes to the oocyst by inheritance through the fertile sex. As a result, reductions in barcode abundance for these sex-specific knock-out mutants often did not reach significance at the B1-MG conversion. Known gene functions in the diploid/polyploid ookinete were also generally not recapitulated in the screen, presumably due to heterozygous rescue as a result of cross-fertilization. While these observations highlight the need for future screens to be designed specifically to reveal sexual and mosquito stage phenotypes, they also rationalize how knock-out alleles of genes functioning in fertility or ookinete/oocyst development can be transmitted to salivary gland sporozoites to reveal additional gene functions after sporozoite transmission to the vertebrate host.

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1312600
Gene Model P. falciparum ortholog PF3D7_1448900
Gene productconserved protein, unknown function
Gene product: Alternative name
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/vector311597
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 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) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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) 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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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