RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4575
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1204800; Gene model (P.falciparum): PF3D7_1006600; Gene product: phosducin-like protein, putative (PhLP3 (previous PhLP1))
PhenotypeNo phenotype has been described
Last modified: 2 January 2019, 15:14
  *RMgm-4575
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) : 30596727
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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/ResearcherKooistra RL, Kanzok SM
Name Group/DepartmentDepartment of Biology
Name InstituteLoyola University Chicago
CityChicago
CountryUSA
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1204800
Gene Model P. falciparum ortholog PF3D7_1006600
Gene productphosducin-like protein, putative
Gene product: Alternative namePhLP3 (previous PhLP1)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) PCR construct 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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMultiple attempts to disrupt PhLP3 were unsuccessful, indicating an essential role during blood stage growth/multiplication.

From the paper:
'We previously identified three putative Trx-like genes in the P. berghei genome as members of the phosducin-like family of proteins (PhLP), specifically PBANKA_1204800, PBANKA_0519700, and PBANKA_1231200. PBANKA_1204800 was designated as PhLP-1 as it was the first PhLP we identified in a Plasmodium species. However, re-analysis of sequence data from multiple organisms suggests that PBANKA_1204800 belongs to the PhLP-3 subgroup, while PBANKA_1231200 is more closely related to the PhLP-1 subgroup. PbPhLP-3 shows the highest conservation among eukaryotes.

A targeting vector was constructed in plasmid pBS-DHFR. A 516 bp fragment comprising the 5’ flanking sequence and a 348 bp fragment of the 3’ flanking sequence of pbphlp-3 were amplified from P. berghei genomic DNA. The 5’ fragment was inserted using the ApaI and HindIII restriction sites immediately upstream of the tgdhfr/ts cassette while the 3’ fragment was inserted into the downstream BamH1 and XbaI restriction sites.

Alignment of recently characterized PhLP-3 homologues, ranging from fungi to mammals, coupled with secondary structure prediction, revealed that PbPhLP-3 consists of an N-terminal helix domain, followed by a thioredoxin-domain and a short C-terminus, an overall organization characteristic for members of the PhLP family.'
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
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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