RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1350
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0108300; Gene model (P.falciparum): PF3D7_0609800; Gene product: palmitoyltransferase DHHC2, putative (DHHC2)
PhenotypeNo phenotype has been described
Last modified: 5 April 2016, 09:51
  *RMgm-1350
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 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26526684
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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/ResearcherSantos JM; Janse CJ; Mair GR
Name Group/DepartmentInstituto de Medicina Molecular
Name InstituteFaculdade de Medicina da Universidade de Lisboa
CityLisbon
CountryPortugal
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0108300
Gene Model P. falciparum ortholog PF3D7_0609800
Gene productpalmitoyltransferase DHHC2, putative
Gene product: Alternative nameDHHC2
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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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAttempts to disrupt the dhhc2 gene in P. berghei were unsuccessful indicating an essential role of DHCC2 for blood stage development/multiplication.

Phenotype analyses of promoter-swap mutants indicate that DHHC2 plays an important role in the development of zygotes into mature ookinetes (see mutants RMgm-1349 and RMgm-1351 for more information on the DHHC2 protein).

To disrupt dhhc2 (PBANKA_010830) we constructed a replacement construct which contains the pyrimethamine resistant tgdhfr/ts as a selectable marker cassette flanked by target sequences for homologous recombination; they were PCR-amplified from P. berghei WT genomic DNA using primers 0739 and 0740, and 0741 and 0742 specific for the 5′ or 3′ flanking regions of the dhhc2 ORF, respectively. The final DNA construct pLIS0065 used for transfection was obtained after digestion of the replacement construct with Asp718I and NotI.
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 1aaaggtaccTTTATTATTTGAGTGTTG
Additional information primer 1g0739
Sequence Primer 2aaaaagcttTTTATATTGATTTTGATTG
Additional information primer 2g0740c
Sequence Primer 3aaagaattcTTGAGTTTATAATATGTC
Additional information primer 3g0741
Sequence Primer 4aaagcggccgcATATCCTAAAAACTATTG
Additional information primer 4g0742c
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