RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5182
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1302300; Gene model (P.falciparum): PF3D7_1438400; Gene product: metacaspase-2 (MCA2, MCA-2)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 25 March 2022, 15:16
  *RMgm-5182
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35264080
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherKumari V, Pandey KC
Name Group/DepartmentICMR-National Institute of Malaria Research
Name InstituteICMR-National Institute of Malaria Research
CityNew Delhi
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-5182
Principal namePbΔPbMCA-2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageIn the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production
Gametocyte/GameteIn the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production
Fertilization and ookineteIn the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production
OocystIn the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production
SporozoiteIn the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Metacaspase-2 (MCA-2) and expresses GFP

Protein (function)
Metacaspases are cysteine-dependent proteases sharing the conserved His-Cys catalytic dyad of caspases (13). These proteases are members of the C14 family, clan CD, with a difference in substrate specificity compared to caspases (14). In addition, metacaspases have a highly acidic S1 pocket leading to arginine and lysine substrate specificity at the P1 position, compared to the aspartic acid specificity reported for caspases.

Three metacaspases have been identified in Plasmodium. MCA1:  PF3D7_1354800, PBANKA_1131400; MCA2 :PF3D7_1354800, PBANKA_1131400; MCA3: PF3D7_1416200, PBANKA_1026500

Phenotype
In the manuscript evidence is presented for: reduced growth of asexual blood stages, reduced gametocyte production (2.7 fold); reduced exflagellation, reduced ookinete formation, ookinete growth arrestment during early development, reduced oocyst production, reduced sporozoite production 

Additional information


Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1302300
Gene Model P. falciparum ortholog PF3D7_1438400
Gene productmetacaspase-2
Gene product: Alternative nameMCA2, MCA-2
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbMCA-2 knockout (ΔPbMCA-2) parasites were generated by a double homologous recombination method. The P. berghei-ANKA wild-type parasites were used to generate ΔPbMCA-2 [PbΔPbMCA-2: Hdhfr & GFP]. In order to generate the PbMCA-2 knockout-targeting construct, a 799 bp fragment was amplified from the 5’ UTR region of PbMCA-2 (gene id: PBANKA_1302300, primers PbMCA-2-5U-XhoI Fw, and PbMCA-2-5U ClaI Rev) and a 741 bp from the 3’UTR of the gene (primers PbMCA-2-3U-NotI Fw and PbMCA-2-3U-AscI Rev). Both UTRs were cloned in pBC derived knockout vector flanking the GFP and DHFR cassette.
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