RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5231
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0516600; Gene model (P.falciparum): PF3D7_1032800; Gene product: leucine-rich repeat protein (PbLRR1)
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 Oocyst; Sporozoite; Liver stage;
Last modified: 29 August 2022, 11:11
  *RMgm-5231
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35920043
MR4 number
top of page
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
top of page
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
top of page
Name of the mutant parasite
RMgm numberRMgm-5231
Principal namePbLRR1KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystA 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.
SporozoiteDespite 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.
Liver stageSporozoites were infective in both in vitro hepatocyte cultures and in mice, albeit with a slightly longer prepatent period.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of LLR1 and 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
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.

Additional information
Analysis of  mutant expressing a C-terminal tagged versions of PbLRR1 (RMgm-5232).the following was shown:
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 

Evidence is presented for an interaction of PbLRR1 to PbPP1c via 3 binding sites

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
top of page
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
top of page
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-232294
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPbLRR1 KO lines were generated by double homologous recombination of a NotI-linearized PlasmoGEM vector. (PbGEM-232294, Wellcome Sanger Institute) transfected into PbGFP ANKA parasites.
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
top of page
  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
top of page
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
top of page
Other details transgene
top of page
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
top of page
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
top of page
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