RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4978
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1017000; Gene model (P.falciparum): PF3D7_1429200; Gene product: AP2 domain transcription factor AP2-O3, putative (ApiAP2; AP2-O3)
Name tag: HA
Phenotype Gametocyte/Gamete; Oocyst;
Last modified: 13 May 2021, 15:50
  *RMgm-4978
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) : 33665945
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherZhenkui C; Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, Schoo
Name InstituteXiamen University
CityXiamen, Fujian
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4978
Principal nameap2-o3::6HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/Gametetagged AP2-O3 expressed in (the nucleus of) female gametocytes (not in males)
Fertilization and ookineteNot different from wild type
Oocysttagged AP2-O3 expressed in mature oocysts (not in ookinetes, early stage oocysts and sporozoites)
SporozoiteNot different from wild type
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses of C-terminal, 6x-HA tagged version of AP2-O3.
The gene has been tagged using CRISPR/cas9 genome editing (see mutant RMgm-1096 for details).

Protein (function)
Most sequence-specific transcription factor families found in other eukaryotes seem to be absent from Plasmodium. Instead, an expansion of a protein family containing one or more apetala2 (AP2) DNA-binding domains was observed across the phylum apicomplexa. In total, 27 members of this family have been found in the human malaria parasite Plasmodium falciparum (although a possible 28th member of the family may be present. In total, 26 of these have syntenic orthologs in rodent malaria species, each with its unique stage-specific expression profile.
From the paper: ' Recently, Modrzynska et al and our group systematically investigated the functions of ApiAP2 TFs in the rodent malaria parasite P. berghei and P. yoelii, respectively (Modrzynska et al, 2017; Zhang et al, 2017b). Both studies revealed that AP2-O3, an ApiAP2 member, is expressed in the gametocytes and essential for the ookinete formation and mosquito transmission (Modrzynska et al, 2017; Zhang et al, 2017b). However, the underlying mechanism is unknown. In this study, we established AP2-O3 as a transcription repressor of the male-associated genes in the female gametocytes, which is essential for maintaining the femalespecific transcriptome.

Phenotype
Tagged AP2-O3 expressed in the nucleus of female gametocytes (not in males) and in mature oocysts (not in ookinetes, early stage oocysts and sporozoites).

Additional information
The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095).

In the paper mutants are described that express mScarlet-tagged AP2-O3, 4xcmyc tagged AP2-O3 and N-terminal, 6x-HA-tagged AP2-O3

Other mutants
see ApiAP2


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1017000
Gene Model P. falciparum ortholog PF3D7_1429200
Gene productAP2 domain transcription factor AP2-O3, putative
Gene product: Alternative nameApiAP2; AP2-O3
Details of the genetic modification
Name of the tagHA
Details of taggingC-terminal
Additional remarks: tagging6x-HA
Commercial source of tag-antibodies
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 modificationCRISPR/Cas9 plasmid pYCm was used for genomic modification (Zhang et al, 2014; Zhang et al, 2017). To construct the plasmids for gene tagging, the C- or N-terminal segments (400–800 bp) of the coding regions were PCR-amplified as the left or right arm and 400–800 bp from 5-UTR or 3-UTR following the translation stop codon as left and right arm, respectively. A DNA fragment encoding GFP, mScarlet, 6HA, or 4Myc was inserted between the left and right arms in frame with the gene of interest. For each gene tagging, at least three sgRNAs were designed to target the C- or N-terminal of the coding region
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