RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-612
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1219500; Gene model (P.falciparum): PF3D7_0709000; Gene product: chloroquine resistance transporter (CRT)
PhenotypeNo phenotype has been described
Last modified: 9 March 2011, 21:16
  *RMgm-612
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 21288823
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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
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Attempts to generate the mutant parasite were performed by
Name PI/ResearcherA. Ecker; V. Lakshmanan, D.A. Fidock
Name Group/DepartmentDepartment of Microbiology and Immunology
Name InstituteColumbia University Medical Center
CityNew York
CountryUSA
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1219500
Gene Model P. falciparum ortholog PF3D7_0709000
Gene productchloroquine resistance transporter
Gene product: Alternative nameCRT
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid HpaI, ScaI
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to select for mutants with a disrupted/deleted crt gene, indicates an essential role of CRT during asexual blood stage development (see also below). In earlier studies in P. falciparum, the failure to disrupt pfcrt (MAL7P1.27) suggested an essential role of CRT for asexual blood stage viability (Waller et al., 2003, J. Biol. Chem. 278, 33593-601).

See RMgm-610 and RMgm-611 for P. berghei mutants in which the crt gene of P. berghei is replaced by the P. falciparum crt gene (MAL7P1.27) of the chloroquine sensitive HB3-strain and chloroquine resistant 7G8-strain, respectively. The analyses of these mutants demonstrate that CRT of P. falciparum can complement the function of CRT of P. berghei.

The knockout/disruption plasmid was constructed in an identical manner to the allelic replacement plasmids described in RMgm-610 and RMgm-611, except that it lacks the pfcrt coding sequence.

PCR analysis detected a minor subpopulation of pbcrt-deleted parasites in 2 of 4 independent transfection experiments, with the majority of the parasites maintaining the knockout plasmid episomally (data not shown). However, the knockout population could not be cloned, indicating that pbcrt is likely required for 'productive' asexual blood stage development.
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 1CTTagatctTTTACTATTTTCAATTTCAACGGTGTG
Additional information primer 1c1 (BglII); 5'targeting region forward
Sequence Primer 2AAGgcgcgcTCCTGTcatatgTAATACACTGATTTAATATATTTAAAAAAATTG
Additional information primer 2c2 (BssHII/NdeI); 5'targeting region reverse
Sequence Primer 3CTTggtaccCTTAAATGATTTTTGTAAATGCCAC
Additional information primer 3c3 (KpnI); 3'targeting region forward
Sequence Primer 4AATaggcctGACGTTATGGCGACGTGTTGCC
Additional information primer 4c4 (StuI); 3'targeting region reverse
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren