RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5232
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0516600; Gene model (P.falciparum): PF3D7_1032800; Gene product: leucine-rich repeat protein (PbLRR1)
Name tag: mCherry
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_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (P230p)
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 29 August 2022, 11:44
  *RMgm-5232
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35920043
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1026
Other information parent lineA drug-selectable marker free reporter line expressing GFP under the constitutive hsp70 promoter
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The mutant parasite was generated by
Name PI/ResearcherFréville A, Khalife J
Name Group/DepartmentCenter for Infection and Immunity of Lille
Name InstituteUniv. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille
CityLille
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-5232
Principal namePbLRR1-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageSee below
Gametocyte/GameteSee below
Fertilization and ookineteSee below
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expressing a C-terminal mCerry-tagged version of PbLRR1 expresses GFP under control of the constitutive hsp70 promoter

Protein (function)
Leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, is one of the most ancient and conserved PP1 interactors.  Protein phosphatase type 1 (or PP1) seems to be responsible for most of the dephosphorylation processes in the parasite.

Phenotype
Most studies on expression of LLR1 were performed with the following mutant:

A C-terminal AID-HA-tagged PbLRR1 line was generated by single homologous recombination. A 1150-bp region of PbLRR1 starting 289 bp downstream from the start codon and lacking a stop codon was inserted into pG362 vector and BsaBI-linearized before transfection into an OsTIR1 expressing pG230 P. berghei ANKA line (both plasmid and parasites are gifts from N. Philip, University of Edinburgh, UK).
 
Analysis of this mutant (PbLRR1-AID-3HA) showed the following:
During the asexual development cycle in the blood, PbLRR1 was undetectable at ring stage but was found expressed in trophozoites at the periphery of the nucleus, and in schizonts as a cytosolic punctuated pattern. PbLRR1 was also observed as cytosolic and perinuclear dots in non-activated gametocytes. Its expression was also detected in activated gametocytes and zygotes (4 h) exhibiting a punctuated perinuclear pattern. By contrast, PbLRR1 was not detectable in ookinetes

Additional information
Analaysis of a mutant lacking expression of LLR1 (5231) showed the following:
- Normal asexual blood stage growth, gametocyte and ookinete production
- A significant decrease (approx. 70%) in the number of oocysts at day 9 post-infection and oocysts at this stage of development were smaller in size. Despite the overall smaller oocyst numbers and sizes in the PbLRR1KO lines, a substantial proportion of oocysts completed development to sporozoites and no significant effects on salivary gland colonization were observed. Sporozoites were infective in both in vitro hepatocyte cultures and in mice, albeit with a slightly longer prepatent period.

Other mutants
  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0516600
Gene Model P. falciparum ortholog PF3D7_1032800
Gene productleucine-rich repeat protein
Gene product: Alternative namePbLRR1
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Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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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 parasiteNo selectable marker
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
Additional remarks selection procedure
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Other details transgene
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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
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3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 PBANKA_0306000
Gene product6-cysteine protein
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren