RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5524
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1019700; Gene model (P.falciparum): PF3D7_1423700; Gene product: NTF2-like domain-containing protein, putative (nuclear transport factor 2-like domain-containing protein)
Phenotype Gametocyte/Gamete;
Last modified: 8 July 2024, 13:12
  *RMgm-5524
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) : 38195464
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/ResearcherNiikura M, Kobayashi F
Name Group/DepartmentDepartment of Environmental Science, School of Life and Environmental Science
Name InstituteAzabu University
CityKanagawa
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5524
Principal nameΔ1019700
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteEvidence is presented for reduced production of male en female gametocytes.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of NTF2-like domain-containing protein, putative

Protein (function)
Export of mRNA through nuclear pores into the cytoplasm is an essential process in all eukaryotic cells. Mature mRNA is recognized by adaptor proteins such as nuclear poly(A) binding protein 2 (NAB2), yeast RNA annealing protein (YRA1), and three serine/arginine-rich (SR) proteins, including nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and hypothetical RNA-binding protein (HRB1). The yeast mRNA export receptor, Mex67/Mtr2, recruited by these adaptor proteins interacts with nuclear pore complex proteins to export mRNA into the cytoplasm. Plasmodium possess orthologs of yeast adaptor proteins, but no orthologs of Mex67 and Mtr2. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain. The NTF2-like domain is necessary for heterodimerization with Mtr2 and for interaction with FG nucleoporins in the nuclear pores. In the PlasmoDB database (www. plasm odb. org/), three proteins containing NTF2-like domains, namely, PBANKA_1019700 (conserved Plasmodium protein), PBANKA_0922900 (mitochondrial import inner membrane translocase subunit TIM44), and PBANKA_1030300 (nuclear transport factor 2), have been annotated. PBANKA_1030300 and PBANKA_0922900 have been reported as being essential to parasite survival during the erythrocytic stage. PBANKA_1030300 binds RanGDP and is associated with protein import to the nucleus from the cytoplasm. PBANKA_0922900 contains a Tim44-like domain, indicating that it localizes to the mitochondria, not to the nucleus of Plasmodium.

Phenotype
Normal development of asexual blood stages. Evidence is presented for reduced production of male en female gametocytes.

Additional information
Aanalysis of a mutant expressing a C-terminal mCherry tagged version of PBANKA_1019700 (RMgm-5525) showed expression in all erythrocytic stages, including trophozoites, schizonts and gametocytes and localization in the cytoplasm of these stages.
To investigate the proteins that interact with the NTF2 domain-containing protein PBANKA_1019700, protein, immunoprecipitation (IP) using anti-mCherry beads was performed and the proteins bound to PBANKA_1019700 were identified through mass spectrometry (MS). Cytoplasmic proteins, including ubiquitin-related proteins (ubiquitin carboxylterminal hydrolase, PBANKA_0210600; ubiquitin-like protein, PBANKA_0823000; HECT-type E3 ubiquitin ligase UT, PBANKA_0802300) and a VPS13 domain-containing protein (PBANKA_1359300) were detected through IP-MS of 1019700::mCherry. However, RNA-binding proteins, such as NAB2 and GBP2, were not detected. In addition, the conserved Plasmodium protein PBANKA_0519900 was detected (see RMgm-5526).

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1019700
Gene Model P. falciparum ortholog PF3D7_1423700
Gene productNTF2-like domain-containing protein, putative
Gene product: Alternative namenuclear transport factor 2-like domain-containing protein
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generated deletion mutants of P. berghei ANKA, the gene-targeting vectors for PBANKA_1019700, PBANKA_1101300 (sbp1) and PBANKA_0519900 were designed and constructed.Briefly, the 5′ and 3′ flanking regions of the open reading frame (ORF) of target genes were amplified by PCR. The PCR products were annealed to either side of the human dihydrofolate reductase (hdhfr)-expressing cassette, the red fluorescent protein gene (mCherry)-hdhfr-expressing cassette or the luciferase gene (luc2)-hdhfr-expressing cassette and amplified by PCR using gene-specific primers. The gene-targeting vectors were introduced into the 5′ and 3′ flanking regions of target genes by double-crossover homologous recombination. To generate transgenic parasites expressing mCherry-fused PBANKA_1019700, the gene-targeting vectors for PBANKA_1019700 were prepared by PCR. The PCR products were annealed to either side of the red fluorescent protein gene (mCherry)-hdhfr-expressing cassette and amplified by PCR using gene-specific primers. The gene-targeting vectors were introduced into the 3′ flanking regions of target genes by double-crossover homologous recombination.
To generate transgenic parasites expressing GFP-fused PBANKA_0519900 and GFP-fused PBANKA_1359300, the gene-targeting vectors for PBANKA_0519900 and PBANKA_1359300 were prepared by PCR, respectively. The PCR products were annealed to either side of the green fluorescent protein gene (gfp)-mutated human deoxyhypusine synthase (hdhps)-expressing cassette and amplified by PCR using gene-specific primers.
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