RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5142

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_1134900; Gene model (P.falciparum): PF3D7_1358600; Gene product: zinc finger protein, putative (Pb103)
Phenotype	Fertilization and ookinete; Oocyst; Sporozoite;

Last modified: 14 January 2022, 14:05

*RMgm-5142

Successful modification	The 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) : 34959491
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. 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/Researcher	Hirai M, Mita T
Name Group/Department	Department of Tropical Medicine and Parasitology, Faculty of Medicine
Name Institute	Juntendo University
City	Tokyo
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-5142
Principal name	Pb103(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Absence of ookinete formation (due to abortion of zygote development).
Oocyst	Absence of ookinete and oocyst formation
Sporozoite	Absence of oocyst and sporozoite formation
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of Pb103 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 Normal, wild type gametocyte/gamete formation. Absence of ookinete formation (due to abortion of zygote development). Absence of oocyst/sporozoite formation. 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. In vitro cross-fertilization experiments were performed between Pb103(-) and CDPK4(-) (male infertility) or Nek4(-) (female infertility) gametes. The self-fertilization of CDPK4(-) and Nek4(-) did not produce ookinetes, while CDPK4(-) x Nek4(-) cross produced ookinetes, which was equivalent to 35.1% of CDPK4(-) female gametes fertilized to Nek4(-) males. Cross-fertilization of Pb103(-) x CDPK4(-) produced ookinetes with slightly lower efficiency compared to CDPK4(-) x Nek4(-) crosses. On the other hand, the Pb103(-) x Nek4(-) cross failed to generate mature ookinetes. These results demonstrate that Pb103 is exclusively required in females. To identify the domains critical for zygote/ookinete development, in this study several transgenic parasites expressing partially deleted Pb103 (RMgm-5143) were generated and assayed for ookinete maturation. 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. Other mutants

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1134900

Gene Model P. falciparum ortholog

PF3D7_1358600

Gene product

zinc finger protein, putative

Gene product: Alternative name

Pb103

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

To generate a construct for Pb103 gene disruption, two PCR fragments covering 500 bp of the 50 and 30 UTRs of the Pb103 gene were amplified by two pairs of primers, Pb03(-)- F1/Pb03(-)-R1 and Pb03(-)- F2/Pb03(-)-R2, respectively, and P. berghei genomic DNA as a template. Then, each PCR product was conjugated to either end of PstI/HindIII digested pL0006 (MRA-775; BEI Resources.) by fusion PCR using primer Pb03(-)-F1/Pb03(-)-R2

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

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