RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1218
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0314200; Gene model (P.falciparum): PF3D7_0217500; Gene product: calcium-dependent protein kinase 1 (CDPK1; PfCPK)
PhenotypeNo phenotype has been described
Last modified: 21 March 2015, 21:47
  *RMgm-1218
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) : 25732065
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/ResearcherGomes AR; Billker O
Name Group/DepartmentWellcome Trust Sanger Institute
Name InstituteWellcome Trust Sanger Institute
CityHinxton Cambridge
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1218
Principal name-
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Gene-deletion mutants were selected after transfection with a single PlasmoGEM vector PbGEM-010677, indicating that PBANKA_031420 is not essential for blood stages.

In this study also evidence was found for successful gene deletion as determined by barcode PCR in a large pool of gene-deletion mutants.

No evidence was found for reduced fitness (growth rate) of blood stages

In this study P. berghei blood stages were transfected with a large pool of barcoded disruption (gene-deletion) vectors. These disruption vectors contained long modification arms to efficiently target the genes of interest.
In addition the vectors contain gene-specific molecular barcodes. Co-transfecting multiple gene-deletion vectors in the same electroporation reproducibly generates complex pools of barcoded P. berghei mutants.
In this study parasites were (mainly) transfected with a pool of vectors targeting protein kinase genes for deletion.
Unsuccessful gene disruption/deletion or successful gene disruption/deletion is determined by the absence or presence of the barcode in the population (as determined by barcode-specific PCR analysis).

To analyze growth curves derived from barcode counting two parameters were considered: (1) the relative abundance of each barcode within the pool, and (2) the relative fitness of each mutant, i.e., the rate at which its abundance changed each day (i.e. growth rate).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0314200
Gene Model P. falciparum ortholog PF3D7_0217500
Gene productcalcium-dependent protein kinase 1
Gene product: Alternative nameCDPK1; PfCPK
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-010677
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