RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5144
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: azami green fluorescent protein (AGFP)
Promoter: Gene model: PBANKA_1134900; Gene model (P.falciparum): PF3D7_1358600; Gene product: zinc finger protein, putative (Pb103)
3'UTR: Gene model: PBANKA_1134900; Gene product: zinc finger protein, putative (Pb103)
Replacement locus: Gene model: PBANKA_1134900; Gene product: zinc finger protein, putative (Pb103)
Transgene
 Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1319500; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (LAP4; LCCL/lectin adhesive-like protein 4; CCp2)
3'UTR: Gene model: PBANKA_1359600; Gene product: transmission blocking target antigen precursor 6-cysteine protein (P48/45)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 14 January 2022, 17:24
  *RMgm-5144
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34959491
MR4 number
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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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The mutant parasite was generated by
Name PI/ResearcherHirai M, Mita T
Name Group/DepartmentDepartment of Tropical Medicine and Parasitology, Faculty of Medicine
Name InstituteJuntendo University
CityTokyo
CountryJapan
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Name of the mutant parasite
RMgm numberRMgm-5144
Principal namePb103-AGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNo fluorescent signal in gametocytes. AGFP fluorescent signal was detected from female gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes.
Fertilization and ookineteNo fluorescent signal in gametocytes. AGFP fluorescent signal was detected from female gametes to ookinetes, while AGFP mRNA was translationally repressed in female gametocytes.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant  expresses azami green fluorescent protein (AGFP) under control of the Pb103 (PBANKA_1134900) promoter and 3'UTR and RFP under control of a female gametocyte/gamete specific promoter (LCCL domain-containing protein)

Protein (function)
- azami green fluorescent protein (AGFP): reporter protein
- promoter: PBANKA_1134900 encodes 870 amino acids with a molecular weight of approximately 103 kDa, and designated it Pb103. InterProScan analysis revealed that Pb103 possesses two CCCH-type (zinc finger) ZF domains. Pb103 is highly conserved among rodent malaria parasites. While orthologous genes are  detected in human malaria parasites with low identity at the entire sequence level, two ZF domains are highly conserved among rodent and human malaria parasites.

Phenotype
No fluorescent signal in gametocytes. AGFP fluorescent signal was detected from female gametes to ookinetes, while AGFP  mRNA was translationally repressed in female gametocytes.

A mutant with the complete Pb103 gene deleted (RMgm-5142) showed normal, wild type gametocyte/gamete formation but absence of ookinete formation (due to abortion of zygote development). 

Additional information
RNAseq data shows that Pb103 is exclusively expressed in female gametocytes. 
Evidence is presented that transcripts are translationally repressed in female gametocytes. Translation occurs after activation of gametocytes.

Other mutants

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameazami green fluorescent protein (AGFP)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe pL1186 plasmid was previously designed to make a transgenic line that expresses azami green (AGFP) and red fluorescent proteins (RFPs) for male and female gametocytes to ookinete, respectively. The pL1186 plasmid was digested with EcoRV/NotI and self-ligated to remove the male-specific promoter and AGFP. The resulting plasmid (female-RFP) was digested with two SacII sites and introduced into the 230p gene locus by double-crossover homologous recombination.
Subsequently, the transgenic parasite clones were subjected to negative selection using 5-fluorocytosine to remove the selectable marker gene. The resulting parasite clone (female-RFP reporter) expresses RFP throughout female gametocytes to ookinetes [35] and was used as a recipient parasite of the Pb103-AGFP plasmid described below. For the construction of the Pb103-AGFP plasmid, fragments covering the 5' untranslated region (UTR) (1.5 kb) and 3' UTR (1.5 kb) of Pb103 and AGFP were amplified using Pb103-50 UTR-F1/Pb103-50 UTR-R1, Pb103-30 UTR-F1/Pb103-30 UTR-R1, and AGFP-F/AGFP-R. These three fragments were serially cloned in the order of Pb103-50 UTR, AGFP, and Pb103-30 UTR into HindIII/PstIdigested pL0006 by an In-Fusion HD cloning kit (TaKaRa). The final plasmid, Pb103-AGFP, was linearized by the PacI site in the 5' UTR of Pb103 and integrated into the recipient parasites by a single crossover. The final transformant, the Pb103-AGFP reporter, was selected by pyrimethamine.
Additional remarks selection proceduresee above
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1134900
Gene Model P. falciparum ortholog PF3D7_1358600
Gene productzinc finger protein, putative
Gene product: Alternative namePb103
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
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3'-UTR
Gene Model of Parasite PBANKA_1134900
Gene productzinc finger protein, putative
Gene product: Alternative namePb103
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_1134900
Gene productzinc finger protein, putative
Gene product: Alternative namePb103
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameRFP
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe pL1186 plasmid was previously designed to make a transgenic line that expresses azami green (AGFP) and red fluorescent proteins (RFPs) for male and female gametocytes to ookinete, respectively. The pL1186 plasmid was digested with EcoRV/NotI and self-ligated to remove the male-specific promoter and AGFP. The resulting plasmid (female-RFP) was digested with two SacII sites and introduced into the 230p gene locus by double-crossover homologous recombination.
Subsequently, the transgenic parasite clones were subjected to negative selection using 5-fluorocytosine to remove the selectable marker gene. The resulting parasite clone (female-RFP reporter) expresses RFP throughout female gametocytes to ookinetes [35] and was used as a recipient parasite of the Pb103-AGFP plasmid described below. For the construction of the Pb103-AGFP plasmid, fragments covering the 5' untranslated region (UTR) (1.5 kb) and 3' UTR (1.5 kb) of Pb103 and AGFP were amplified using Pb103-50 UTR-F1/Pb103-50 UTR-R1, Pb103-30 UTR-F1/Pb103-30 UTR-R1, and AGFP-F/AGFP-R. These three fragments were serially cloned in the order of Pb103-50 UTR, AGFP, and Pb103-30 UTR into HindIII/PstIdigested pL0006 by an In-Fusion HD cloning kit (TaKaRa). The final plasmid, Pb103-AGFP, was linearized by the PacI site in the 5' UTR of Pb103 and integrated into the recipient parasites by a single crossover. The final transformant, the Pb103-AGFP reporter, was selected by pyrimethamine.
Additional remarks selection proceduresee above
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1319500
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP4; LCCL/lectin adhesive-like protein 4; CCp2
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
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3'-UTR
Gene Model of Parasite PBANKA_1359600
Gene producttransmission blocking target antigen precursor 6-cysteine protein
Gene product: Alternative nameP48/45
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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
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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren