RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5143
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1134900; Gene model (P.falciparum): PF3D7_1358600; Gene product: zinc finger protein, putative (Pb103)
Details mutation: Pb103 with several deletions of (zinc finger) domains
Phenotype Fertilization and ookinete;
Last modified: 14 January 2022, 15:35
  *RMgm-5143
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34959491
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/ResearcherHirai M, Mita T
Name Group/DepartmentDepartment of Tropical Medicine and Parasitology, Faculty of Medicine
Name InstituteJuntendo University
CityTokyo
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5143
Principal namePb103(mutated)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteDeleting either of two zinc finger (ZF) domains (ZFs) but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
Several mutants were generated with a mutated Pb103 with deletions of either zinc finger domains (ZF's) or the C-terminal region.

To identify crucial domains for ookinete maturation, four Pb103 deletion mutants were made and designated them -ZF1, -ZF2, -C215, and -C495. The -ZF-1 or -ZF-2 lacks the first or second ZF domain, while -C215 or -C495 lacks the respective numbers of amino acids from the C-terminus. Mutants were generated using CRIPR/Cas gene editing as described for mutant RMgm-5042.

Protein (function)
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
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). 

To identify crucial domains for ookinete maturation,  four Pb103 deletion mutants were made and designated them -ZF1, -ZF2, -C215, and -C495. The -ZF-1 or -ZF-2 lacks the first or second ZF domain, while -C215 or -C495 lacks the respective numbers of amino acids from the C-terminus.

Deleting either of two zinc finger (ZF) domains (ZFs) but not the C-terminal region abolished zygote/ookinete development, highlighting the indispensable roles of ZFs in parasite sexual development, most likely via translational repression.

Additional information
RNAseq data shows that Pb103 is exclusively expressed in female gametocytes. 
Evidence is presented that transcripts are translationally repressed in female gametocytes (RMgm-5144). Translation occurs after activation of gametocytes.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1134900
Gene Model P. falciparum ortholog PF3D7_1358600
Gene productzinc finger protein, putative
Gene product: Alternative namePb103
Details of the genetic modification
Short description of the mutationPb103 with several deletions of (zinc finger) domains
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 modificationFour Pb103 partial deletion mutants were generated by using CRISPR/Cas9. For the construction of -C215, two fragments covering 455 to 655 a.a. (Frag. A) and 0.5 kb of 30 UTR (Frag. B) were amplified by two pairs of primers: -C215-F/-C215-R and -C215-3UTR-F/-C215-3UTR-R. These two fragments were serially inserted as a donor into AflII/HindIII of pBS-1, which was a CRISPR/Cas9 plasmid customized to P. berghei (see mutant RMgm-5042). For the -C495 mutant, two fragments covering 189–375 a.a. (Frag. C) and 0.5 kb of 30 UTR (Frag. D) were amplified by two pairs of primers: -C495-F/-C495-R and -C495-3UTRF/-C495-3UTR-R. These two fragments were inserted into pBS-1, as described above. For the -ZF1 mutant, fragments covering the 50 UTR (0.5 kb) to the first 10 a.a. (Frag. E) and 38 to 220 a.a. motif (Frag. F) were amplified using -ZF1-F1/-ZF1-R1 and -F1-F2/-ZF1-R2. For the -ZF2 mutant construct, fragments covering the 50 UTR (0.2 kb) to the first 79 a.a. (Frag. G), and 115 a.a. to 270 a.a. (Frag. H) were amplified using -ZF2-F1/-ZF2-R1 and -ZF2-F2/-ZF2-R2, respectively. The fragment, E/F, and G/H were inserted as donors into pBS-1. The PAM sites were searched using CHOPCHOP (http://chopchop.cbu.uib.no). The double-stranded DNA coding for gRNA was inserted into the BsmBI site in pBS-1. The resultant plasmids were introduced into P. berghei, and transformants were cloned. All clones were subjected to Sanger sequencing to confirm that the expected regions in the Pb103 gene were deleted.
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