RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1456
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PBANKA_0833000; Gene model (P.falciparum): PF3D7_0932200; Gene product: profilin (PFN; PRF)
Promoter: Gene model: PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
3'UTR: Gene model: PBANKA_0833000; Gene product: profilin, putative (PFN; PRF)
Insertion locus: Gene model: PBANKA_0833000; Gene product: profilin, putative (PFN; PRF)
Phenotype Sporozoite; Liver stage;
Last modified: 17 June 2016, 19:04
  *RMgm-1456
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) : 27226484
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 Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherSato Y; Matuschewski K
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-1456
Principal nameprofilin(CSP)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNo salivary gland sporozoites
Liver stageNo invasion of hepatocytes (in vitro) by hemocoel sporozoites.
No infection of mice by mosquito bite.
Additional remarks phenotype

Mutant/mutation
The mutant contains an additional copy of the profilin gene under the control of the csp promoter. The profilin gene is N-terminal tagged with a FLAG tag

Protein (function)
Profilins are ubiquitous and essential actin monomer binding proteins, known to interact with proline-rich regions in a variety of proteins and to regulate actin filament formation. For fast actin polymerization at selected sites, profilin is an essential control element by recruiting actin monomers in a polymerizable form to actin polymerization machineries. Profilins are usually defined by their shared, highly conserved structure and by their ability to bind actin, proline-rich sequences.

Phenotype
In this study three mutants are generated and analysed that contain an additional copy of the profilin gene under the control of three different promoters (ama-1, ctrp and csp). See also mutants RMgm-1455 and RMgm-1457

No phenotype was observed in blood stages

The profilin(CTRP) produced normal numbers of ookinetes with normal motility and normal oocyst production

In the profilin(AMA1) mutant a strong reduction in salivary gland sporozoites was observed (2,000-5,000 salivary gland sporozoites, a two- to five-fold reduction as compared to WT sporozoites).
In the profilin(CSP) no salivary gland sporozoites were observed. Profilin(AMA1)  sporozoites showed a reduced number of trails compared to WT, but the majority was able to perform productive motility.
Profilin(CSP) sporozoites were able to attach to hepatoma cells in a similar manner as other transgenic and WT parasites, despite their incapacity to perform gliding locomotion.
In  agreement with the observed lack to colonize salivary glands and perform gliding motility, profilin(CSP) sporozoites were also unable to invade hepatoma cells.
No infection of mice by bite of profilin(CSP) infected mosquitoes.
Reduced in vitro invasion of hepatocytes by profilin(AMA1) sporozoites (but normal prepatent period after mosquito infection of mice)

Additional information

Other mutants
See PF3D7_0932200 for other 'profilin' mutants

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PBANKA_0833000
Gene Model P. falciparum ortholog PF3D7_0932200
Gene productprofilin
Gene product: Alternative namePFN; PRF
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 modificationThe construct was integrated into the profilin gene locus by single cross-over integration resulting in allele duplication with one WT profilin copy and one copy under the control of the stage-specific promoter.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
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_0833000
Gene productprofilin, putative
Gene product: Alternative namePFN; PRF
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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_0833000
Gene productprofilin, putative
Gene product: Alternative namePFN; PRF
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