RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4864
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0902900; Gene model (P.falciparum): PF3D7_1146100; Gene product: conserved protein, unknown function (PH-like (PHL) domain-containing protein; PHL4)
Name tag: GFP
Phenotype Fertilization and ookinete;
Last modified: 1 September 2020, 15:08
  *RMgm-4864
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32736136
MR4 number
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).
The mutant parasite was generated by
Name PI/ResearcherTremp AZ, Dessens JT
Name Group/DepartmentDepartment of Infection Biology, Faculty of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene & Tropical Medicine
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4864
Principal namePHL4-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookinetePHL4-GFP located in the crystalloid of ookinetes
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of PHL4

Protein (function)
The protein PHL4 was identified in a GFP-affinity pulldown with high accuracy mass spectrometry using the GFP-tagged ookinete crystalloid protein LAP3 (PBANKA_0204500).

See below for more information on the protein

Phenotype
PHL4-GFP located in the crystalloid of ookinetes

Additional information
From the paper: 
The analysis also identified PBANKA_0704900, a putative pleckstrin homology (PH) domain-containing protein whose orthologue in P. yoelii was recently shown to reside in the crystalloids. Using this protein's amino acid sequence in BLAST homology searches, we identified three paralogues in the Plasmodium genome, namely PBANKA_0417200, 0902900 and 0704800. These were also present in the LAP3 interactome. The genes for two of these proteins are in fact located tandemly on chromosome 7, pointing to a relatively recent gene duplication event. Their shared domain only has weak homology with the archetypal PH domain, and in fact contains a unique amino acid signature C(X)9W(X)9C (one letter amino acid code, X=any amino acid). We therefore propose the name PH-like (PHL) domain-containing proteins for this group of molecules. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0902900
Gene Model P. falciparum ortholog PF3D7_1146100
Gene productconserved protein, unknown function
Gene product: Alternative namePH-like (PHL) domain-containing protein; PHL4
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAn approximately 1.5 kb fragment corresponding to the coding sequence and 5’UTR of PBANKA_0417200 was PCR amplified from P. berghei genomic DNA with primers PH1-F (TTGGGCTGCAGTCGAGGTACCACAAAACAATTGTCATAAAATAGTTCTTG) and PH1-R (ATGAGGGCCCCTAAGCTCATATCGTTATCGTTTTCTTCATTG) and cloned into SalI/HindIII-digested plasmid pBS-EGFP-hDHFR to give pBS-PH1/GFP. The plasmid was linearized with HindIII before gene targeting by single crossover homologous recombination. An approximately 1.8 kb fragment corresponding to the coding sequence and 5’UTR of PBANKA_0704800 was PCR amplified from P. berghei genomic DNA with primers PH2-F (TTGGGCTGCAGTCGAGGTACCATGCGCATTTATAATATACATAAATAAG) and PH2-R (ATGAGGGCCCCTAAGCTCAAATTATCATCATCATTATCTTCATATTCTTC) and cloned into SalI/HindIII-digested plasmid pBS-EGFP-hDHFR to give pBS-PH2/GFP. The plasmid was linearized with ClaI before gene targeting
by single crossover homologous recombination.
An approximately 2.0 kb fragment corresponding to the coding sequence and 5’UTR of PBANKA_0704900 was PCR amplified from P. berghei genomic DNA with primers PH3-F (TTGGGCTGCAGTCGAGGTACCATTTCTTATTAATAGACAAAACAAAAATAAT) and PH3-R (ATGAGGGCCCCTAAGCTCTTAAGAGAAATATTTGGATTACTGCTTTT) and cloned into SalI/HindIII-digested plasmid pBS-EGFP-hDHFR to give pBS-PH3/GFP. The plasmid was linearized with NheI before gene targeting by single crossover homologous recombination.
An approximately 1.8 kb fragment corresponding to the coding sequence and 5’UTR of PBANKA_0902900 was PCR amplified from P. berghei genomic DNA with primers PH4-F (TTGGGCTGCAGTCGAGGTACCTTTGTACATACATTCAAAAGGCG) and PH4-R (ATGAGGGCCCCTAAGCTGGTCTCTGCTTTTATGGAAACTAAAAAA) and cloned into SalI/HindIII-digested plasmid pBS-EGFP-hDHFR to give pBS-PH4/GFP. The plasmid was linearized with ClaI before gene targeting by single
crossover homologous recombination.
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