RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-562
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0933000; Gene model (P.falciparum): PF3D7_1114700; Gene product: serine/threonine protein kinase, putative (prk4)
PhenotypeNo phenotype has been described
Last modified: 21 December 2011, 14:16
  *RMgm-562
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) : 20951971
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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 P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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Attempts to generate the mutant parasite were performed by
Name PI/ResearcherR. Tewari, O. Billker
Name Group/DepartmentUniversity of Nottingham
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0933000
Gene Model P. falciparum ortholog PF3D7_1114700
Gene productserine/threonine protein kinase, putative
Gene product: Alternative nameprk4
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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
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption nearly complete
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene has been targeted for gene deletion using a construct aimed at integration into the genome by double cross-over homologous recombination

The gene has been targeted for disruption in a 'kinome-wide' study for deletion of genes encoding Plasmodium protein kinases (protein kinase-like proteins).

See the paper for additional information

Disruption of the P. falciparum ortholog has been attempted (Solyakov et al., 2011, Nat Commun, 2:565).
The gene is likely essential for asexual proliferation. After transfection with a KO vector a weak PCR signal diagnostic for integration was observed, indicating that integration does transiently occur but parasites with a disrupted locus do not persist. Cloning will be required to validate this interpretation for this gene.
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 1CCCCGGGCCCCTAAGCGCTCAAAAAGTGACCAC
Additional information primer 1primer sequence target region 1a
Sequence Primer 2GGGGAAGCTTCGTACATCTCTCTCAAAGTTGAAAAATATG
Additional information primer 2primer sequence target region 1b
Sequence Primer 3CCCCGAATTCGATGTATGGTCTGCAGCAGCCACTG
Additional information primer 3primer sequence target region 2a
Sequence Primer 4GGGGTCTAGAGGATGTTGCAATGCTTGATCTGG
Additional information primer 4primer sequence target region 2b
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: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren