RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5140
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0808100; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
Name tag: 3xFlag
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: PY17X_0811300; Gene product: 6-cysteine protein (B9)
Phenotype Sporozoite; Liver stage;
Last modified: 7 January 2022, 16:59
  *RMgm-5140
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) Not published (yet)
MR4 number
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 lineThe mutant expresses GFP under the control of the HSP70 promoter. The GFP gene is introduced into the silent 230p locus using the GOMO method of transfection
The mutant parasite was generated by
Name PI/ResearcherFernandes P, Silvie O
Name Group/DepartmentSorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses
Name InstituteCIMI-Paris
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-5140
Principal namePbB9-Flag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNormal/wild-type sporozoite development and infectivity, indicating that the 3xFlag epitope had no detrimental effect on B9 function.
B9-Flag detected in salivary gland sporozoites, with a distribution pattern typical of a micronemal protein. It distributes in numerous vesicles localized on each side of the nucleus. B9 was detected in Western analysis as a single band around 80 kDa. Upon stimulation of microneme secretion, B9 was also recovered in the supernatant fraction as a slightly smaller band, indicating that B9 is secreted from sporozoites upon activation, possibly after proteolytic processing. B9 was not detected on the surface of sporozoites by immunofluorescence, irrespective of parasite activation, suggesting that following microneme secretion, B9 is mainly released as a shed protein.
Liver stageNormal/wild-type sporozoite development and infectivity, indicating that the 3xFlag epitope had no detrimental effect on B9 function.
Additional remarks phenotype

Mutant/mutation
The mutant expression a C-terminal 3xFLAG-tagged version of B9 and expresses GFP under the control of the HSP70 promoter. The b9 gene is tagged in mutant RMgm-1026 that expresses GFP under the control of the HSP70 promoter. The GFP gene is introduced into the silent 230p locus using the GOMO method of transfection.

Because B9 is predicted to be glycosylphosphatidylinositol (GPI) anchored, the 3xFlag tag was inserted towards the C-terminus of the protein, downstream of the putative 6-cys domains but upstream of the predicted omega site (aspartate residue at position 826). 

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.10.25.465731

Protein (function)
The B9 protein contains a 4-cysteine domain with a structure that is highly similar to the structure of 4-cysteine domains in the known proteins of the 6-Cys family and is identified as a 6-Cys-related protein. B9 is predicted to be glycosylphophatidylinositol (GPI) anchored. 

Phenotype
Normal/wild-type sporozoite development and infectivity, indicating that the 3xFlag epitope had no detrimental effect on B9 function. 
B9-Flag detected in salivary gland sporozoites, with a distribution pattern typical of a micronemal protein. It distributes in numerous vesicles localized on each side of the nucleus. B9 was detected in Western analysis as a single band around 80 kDa. Upon stimulation of microneme secretion, B9 was also recovered in the supernatant fraction as a slightly smaller band, indicating that B9 is secreted from sporozoites upon activation, possibly after proteolytic processing. B9 was not detected on the surface of sporozoites by immunofluorescence, irrespective of parasite activation, suggesting that following microneme secretion, B9 is mainly released as a shed protein.

Additional information
B9 did not co-localize with the apical membrane antigen 1 (AMA1), suggesting that the two proteins are present in distinct microneme subsets in salivary gland sporozoites

Evidence in the paper is presented that: 
- B9 is required for sporozoite invasion
- B9 is secreted from the sporozoite micronemes
- B9 contains a CyRPA-like beta propeller domain, required for B9 function
- The propeller domain of B9 interacts with P36 and P52

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0808100
Gene Model P. falciparum ortholog PF3D7_0317100
Gene product6-cysteine protein
Gene product: Alternative nameB9
Details of the genetic modification
Name of the tag3xFlag
Details of taggingC-terminal
Additional remarks: taggingBecause B9 is predicted to be glycosylphosphatidylinositol (GPI) anchored, the 3xFlag tag was inserted towards the C-terminus of the protein, downstream of the putative 6-cys domains but upstream of the predicted omega site (aspartate residue at position 826)
Commercial source of tag-antibodies
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
Promoter of the selectable markerunknown
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

  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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThe 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
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_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 PY17X_0811300
Gene product6-cysteine protein
Gene product: Alternative nameB9
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