RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1251
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0712900; Gene model (P.falciparum): PF3D7_0817900; Gene product: high mobility group protein B2 (HMGB2)
Phenotype Asexual bloodstage;
Last modified: 8 May 2015, 18:28
  *RMgm-1251
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) : 25916985
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/ResearcherBriquet S, Vaquero C
Name Group/DepartmentSorbonne Universités
Name InstituteCentre d'Immunologie et des Maladies Infectieuses (CIMI-Paris)
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-1251
Principal nameΔhmgb2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageEvidence is presented for normal growth of blood stages but reduction of cerebral complications in C57Bl6 mice that are susceptible to ECM (Experimental Cerebral Malaria).
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of HMGB2

Protein (function)
High mobility group box (HMGB) proteins are nuclear proteins originally shown to be loosely associated with DNA and to participate, to at least some extent, in the regulation of gene transcription. Subsequent studies have shown that the human HMGB proteins composed of two HMGB domains, Box A and Box B in tandem and in particular the HMGB1 isoform are actively secreted from activated innate immune cells, namely macrophages and can be released from injured cells as well. In humans, HMGB1 has an role as extracellular soluble protein that signals tissue injury and initiates inflammatory responses.
Plasmodium species have two high mobility group proteins, HMGB1 and HMGB2, which consist of only one HMGB domain also comprising a 20 amino acid peptide potentially bearing pro-inflammatory activity.

See also RMgm-162 for a P. yoelii mutant lacking expression of HMGB2. These parasites showed a slightly reduced growth rate of blood stages and reduced ookinete and oocyst production.

Phenotype
Evidence is presented for normal growth of blood stages but reduction of cerebral complications in C57Bl6 mice that are susceptible to ECM (Experimental Cerebral Malaria).

Additional information

Other mutants
See also RMgm-162 for a P. yoelii mutant lacking expression of HMGB2. These parasites showed a slightly reduced growth rate of blood stages and reduced ookinete and oocyst production.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0712900
Gene Model P. falciparum ortholog PF3D7_0817900
Gene producthigh mobility group protein B2
Gene product: Alternative nameHMGB2
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTwo PCR fragments flanking the HMGB2 open reading frame (ORF) were amplified from the genomic DNA using oligonucleotides listed in Supplementary Table S1. Amplification of the 5’ untranslated region (UTR) with the primer combination 5’-hmgb2-for and 5’-hmgb2-rev including ApaI and SmaI restriction sites, respectively resulted in a 526-bp fragment and was cloned upstream to the positive selection marker human dihydrofolate reductase (hudhfr) previously introduced into the pBC SK- vector (Stratagene) under the control of EF1α promoter and of dhrf/ts 3’UTR (dihydrofolate reductase/thymidylate synthase). Next, the 3’UTR region was amplified with 3’-hmgb2-for and 3’-hmgb2-rev primers including NotI and AscI restriction sites. This fragment was inserted downstream to the hudhrf box resulting in the hmgb2 targeting vector pBC-5’B2hudhfr3’B2 allowing replacement of the endogenous hmgb2 locus in PbANKA upon a double cross-over homologous recombination and subsequent selection with the antifolate pyrimethamine. P. berghei parasite transfections were performed with 5 μg ApaI/AscI-digested pBC-5’B2hudhfr3’B2.
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