RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4840
Malaria parasiteP. chabaudi
Genotype
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: PCHAS_0308200; Gene product: 6-cysteine protein P230p, putative (P230p)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Oocyst; Sporozoite;
Last modified: 14 August 2020, 14:43
  *RMgm-4840
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 32500098
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. chabaudi
Parent strain/lineP. c.chabaudi AS
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherMarr EJ, Thompson J
Name Group/DepartmentInstitute of Immunology and Infection Research, School of Biological Sciences
Name InstituteUniversity of Edinburgh
CityEdinburgh
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4840
Principal namePcAS-GFP(ML)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGFP expression
Gametocyte/GameteGFP expression in gametocytes
Fertilization and ookineteNot tested
OocystGFP expression
SporozoiteGFP expression
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under control of the constitutive and strong P. berghei hsp70 promoter and is drug-selectable marker free. The selectable marker cassette has been removed by negative selection resulting in a drug-selectable marker free parasite line.

Protein (function)

Phenotype
GFP expression in blood stages, oocysts and sporozoites (liver stages not tested).
Fluorescent PcAS parasites transmit through the mosquito and show wild-type infection dynamics.

Additional information
To generate stable, marker-free fluorescent P. chabaudi mother-lines, plasmids pCAT230p-G6 and pCAT230p-M6 were transfected into P. chabaudi AS parasites according to this modified protocol to introduce GFP and mCherry into the P. chabaudi 230p silent locus by double cross-over recombination. After pyrimethamine selection of PcAS-GFP.Δ230p and PcASmCh.Δ230p parasites and genotypic verification, PcAS-GFP.Δ230p–infected mice were provided with 5-FC to select parasites that had lost the drug-selectable cassettes by homologous recombination of pbdhfr-ts 3’utr duplicate sequences. The resulting marker-free fluorescent parasites were cloned by limiting dilution to produce the mother-line, PcAS-GFPML (RMgm-4840).

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationpCAT-230p-G6 was generated by replacing the sil6 target regions of pBAT-SIL6-G6 (Kooij et al., 2012; RMgm-757)) with bps 640-1680 and 3312-4255 of the P. chaubaudi 230p (PCHAS_0308200) locus. The 230p target regions were amplified from P.c. chabaudi AS genomic DNA using primers 5’230pF x 5’230p (5’ target region) and 3’230pF x 3’230pR (3’ target region) and cloned into the multiple cloning sites of pBAT-Sil6-G6 or –M6 to generate pCAT-230p-G6 or pCAT-230p–M6.
Additional remarks selection procedureThe selectable marker cassette has been removed by negative selection resulting in a drug-selectable marker free parasite
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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
3'-UTR
Gene Model of Parasite PBANKA_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 PCHAS_0308200
Gene product6-cysteine protein P230p, putative
Gene product: Alternative nameP230p
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