RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4983
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0805800; Gene model (P.falciparum): PF3D7_0705400; Gene product: DNA replication licensing factor MCM7 (mcm7)
Name tag: GFP
TaggedGene model (rodent): PY17X_1323300; Gene model (P.falciparum): PF3D7_1455800; Gene product: LCCL domain-containing protein (CCp2, LAP4)
Name tag: mCherry
Phenotype Gametocyte/Gamete;
Last modified: 13 May 2021, 16:17
  *RMgm-4983
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, 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 RMgm-4475
Other information parent lineThe parent mutant line RMgmDB-4475 expresses a C-terminal mCherry-tagged version of CCp2. The mutant does not contain a drug-selectable marker
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-4983
Principal namemcm7::gfp (DTS4)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteAll the 4 GFP-fusion proteins were expressed and nuclear localized in the male gametocytes, but were not detectable (Dpod2::GFP and Dpod1::GFP) or present at extremely low abundance (Rpa1::GFP and Mcm7::GFP) in the female gametocytes.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses of C-terminal, GFP-tagged version of mcm7 and a C-terminal, mCherry-tagged version of CCp2, LAP4.
The gene has been tagged using CRISPR/cas9 genome editing (see mutant RMgm-1096 for details).

Protein (function)

Phenotype
Protein expression of upregulated male genes in the AP2-O3 null female gametocytes
From the paper: ' We selected 4: dpod2 (PY17X_0408600, DNA polymerase delta small subunit), dpod1 (PY17X_0502300, DNA polymerase delta catalytic subunit), rpa1 (PY17X_0419400, replication protein A1 small fragment), and mcm7 (PY17X_0805800, DNA replication licensing factor). Each of these 4 genes was endogenously tagged with a gfp-coding sequence at the C-terminus in the female-identity reporter strain ccp2::mCherry, yielding 4 double-tagged strains, including ccp2::mCherry;dpod2::gfp (DTS1), ccp2::mCherry;dpod1::gfp (DTS2), ccp2::mCerry;rpa1::gfp (DTS3), and ccp2::mCherry;mcm7::gfp (DTS4).
All the 4 GFP-fusion proteins were expressed and nuclear localized in the male gametocytes , but not were detectable (Dpod2::GFP and Dpod1::GFP) or present at extremely low abundance (Rpa1::GFP and Mcm7::GFP) in the female gametocytes. Next, we removed ap2-o3 gene in each individual DTS strain. As expected, AP2-O3 disruption (see RMgm-4979) did not significantly affect the level of any of these 4 proteins in the male gametocytes as revealed by both fluorescence microscopy and flow cytometry In clear contrast, protein levels of Dpod2::GFP and Dpod1::GFP in the female gametocytes were dramatically increased, while Rpa1::GFP and Mcm7::GFP levels in the female gametocytes showed a modest increase compared to the corresponding parental strains.'

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0805800
Gene Model P. falciparum ortholog PF3D7_0705400
Gene productDNA replication licensing factor MCM7
Gene product: Alternative namemcm7
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1323300
Gene Model P. falciparum ortholog PF3D7_1455800
Gene productLCCL domain-containing protein
Gene product: Alternative nameCCp2, LAP4
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor tagging CCp2 the CRISPR/Cas9 method was used as described for mutants RMgm-1095 and RMgm-4071.
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