RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4725
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: Argonaute 2 (Argo2)
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: Not available; Gene product: Not available
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
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 (230p)
Phenotype Asexual bloodstage; Oocyst; Sporozoite;
Last modified: 9 February 2020, 15:54
  *RMgm-4725
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31680162
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-7
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherHentzschel F, Grimm D
Name Group/DepartmentCenter for Infectious Diseases / Parasitology
Name InstituteHeidelberg University Hospital
CityIm Neuenheimer Feld
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4725
Principal namePbAgo2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageAgo2 expression in blood and liver stages. (Slightly)Reduced growth of blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystReduced oocyst formation
SporozoiteReduced oocyst and sporozoite formation
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses a FLAG tagged version of Argonaute 2 (Argo2) under control of the constitutive hsp70 promoter. In addition it expresses GFP under control of the constitutive eef1a promoter.
The Argonaute 2 construct is integrated by double cross-over integration in a locus on P. berghei chromosome 6 (Sil6 locus) between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220).
The mutant is drug-selectable marker free. Introduction of the hsp70-Argonaute 2 expression cassette is performed using positive selection. After cloning of the transgenic parasites the drug-selectable marker cassette is removed by applying negative selection, resulting in selection of parasites that have removed the positive/negative selectable marker hdhft/yfcu by homologous recombination.

This mutant has been used to express specific AgoshRNA's and evidence is provided for (partly) gene-silencing.

Protein (function)
In most eukaryotes, a tool for gene-siliencing is available in the form of RNA interference (RNAi), an endogenous pathway for gene regulation on the mRNA level that can be usurped by using small interfering or short hairpin RNAs (siRNAs or shRNAs, respectively) as exogenous RNAi triggers. However, Plasmodium species lack the canonical RNAi machinery, including the key enzymes Dicer that processes transcribed shRNAs into siRNAs, as well as Argonaute 2 (Ago2) that, when loaded with the siRNA, binds and cleaves target mRNA. Interestingly, though, a noncanonical RNAi pathway has recently been described in mammalian cells that requires only Ago2 to process a special type of shRNAs. These so-called AgoshRNAs have a shorter stem and loop than conventional shRNAs, which prevents their recognition by Dicer and instead facilitates direct loading into, and processing by, Ago2. The resulting protein-RNA complex then binds to a complementary target mRNA and causes its cleavage and degradation. The lack of endogenous RNAi machinery in the malaria parasite Plasmodium hampers gene annotation and hence antimalarial drug and vaccine development. Here, we engineered rodent Plasmodium berghei to express a minimal, non-canonical RNAi machinery that solely requires Argonaute 2 (Ago2) and a modified short hairpin RNA, so-called.
AgoshRNA.

Phenotype
Expression of Ago2 in blood and liver stages is shown. The mutant had (slightly) reduced growth of blood stages and produced decreased numbers of oocysts and sporozoites.

This mutant has been used to express specific AgoshRNA's and evidence is provided for (partly) gene-silencing.

Additional information
We found remarkable differences between the transcriptome of blood stages of the parental PbGFPcon and PbAgo2 (with or without AgoshRNA).  Specifically, in PbAgo2, we observed a significantly lower abundance of female gametocyte-specific transcripts that prior work had shown to be down-regulated upon deletion of DOZI.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameArgonaute 2 (Argo2)
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 modificationThe construct is integrated by double cross-over integration in a locus on P. berghei chromosome 6 between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220), both displaying only limited gene expression in the stages studied thus far.
Additional remarks selection procedureIntroduction of the hsp70-Argonaute 2 expression cassette is performed using positive selection. After cloning of the transgenic parasites the selectable marker cassette is removed by applying negative selection, resulting in selection of parasites that have removed the positive/negative selectable marker hdhft/yfcu by homologous recombination
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 Not available
Gene productNot available
Gene product: Alternative name
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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