RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5102
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4, up-regulated in infective sporozoites, ETRAMP10.3)
Details mutation: Pbuis4 gene replaced with P. falciparum etramp8 gene (PF3D7_0829600)
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 Liver stage;
Last modified: 8 November 2021, 17:02
  *RMgm-5102
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34715088
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 P. berghei ANKA 507cl1 (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).
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The mutant parasite was generated by
Name PI/ResearcherBrandsma AM, Montagna GN
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-5102
Principal nameuis4::Pfetramp8
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageStrongly reduced liver stage development (comparable to mutants lacking expression of P. berghei uis4). No blood-stage infection after infection of mice by mosquito bite or intravenous injection of sporozoites.
Additional remarks phenotype

Mutant/mutation
In the mutant the uis4 gene has been replaced by the P. falciparum etramp8 gene (PF3D7_0829600). This gene is under control of the 3' and 5' UTR regions of uis4 of P. berghei. In addition, the mutant expresses GFP under control of the constitutive eef1a promoter

Protein (function)
UIS4  is expressed throughout liver stage development and localizes to the parasitophorous vacuole. UIS4 has a total length of 220 amino acid residues and can be divided into three domains: an amino-terminal domain of 54 residues including the signal peptide encompassing 25 residues, the trans-membrane region and a positively charged carboxy-terminal domain of 143 residues. Inside an infected hepatocyte, UIS4 is distributed at the PVM and the tubovesicular network (TVN), which forms vesicular protrusions into the host cell cytoplasm.

Phenotype
Strongly reduced liver stage development (comparable to mutants lacking expression of P. berghei uis4). No blood-stage infection after infection of mice by mosquito bite or intravenous injection of sporozoites. 

Additional information
From the Abstract:
'Early transcribed membrane proteins (ETRAMPs) form a unique protein family in malaria parasites. These molecules are expressed during Plasmodium intracellular phases and inserted at the parasite parasitophorus vacuole membrane (PVM), which constitutes the host-parasite interface. Upregulated in infectious sporozoites 4 (UIS4) is an essential ETRAMP of liver stages of the murine malaria model parasite Plasmodium berghei.We generated transgenic P. berghei parasites where UIS4 was replaced by Plasmodium falciparum ETRAMP8 or ETRAMP10.3. Both ETRAMPs were expressed in transgenic parasite lines, but liver stage maturation was impaired, indicating that the selected ETRAMPs failed to substitute the function of UIS4. As a control, we included the UIS4 ortholog from the murine parasite Plasmodium chaubaudi. We observed that PcUIS4 successfully restores UIS4 function in P. berghei'

PfETRAMP8, PfETRAMP10.3 and PfETRAMP14.2 are the only members of the P. falciparum ETRAMP family expressed in sporozoites. In contrast to UIS4, PfETRAMP10.3 is also expressed in blood stages and is considerably shorter in amino acid length (Table 1). Previous work demonstrated that PfETRAMP10.3 did not complement the function of UIS4 in P. yoelii parasites. 

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites, ETRAMP10.3
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Details of the genetic modification
Short description of the mutationPbuis4 gene replaced with P. falciparum etramp8 gene (PF3D7_0829600)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn order to generate the trans-species complementation parasites, we amplified the 5’UTR of PbUIS4 gene and fused it to the ETRAMP8 or ETRAMP10.3 ORF, respectively, by PCR. We used a first step PCR cycle of 95°C, 5’ and 60°C 5' for the fragment annealing, followed by addition of the appropriate primers and 34 cycles of PCR under standard conditions (primers are listed in Supplementary Table S1). The fragments containing the PbUIS4 5’UTR fused to the corresponding PfETRAMP ORF were cloned into a B3D+ vector (Silvie et al., 2008). Next, the PbUIS4 3’UTR was added to facilitate the integration of the constructs in the PbUIS4 locus by a double recombination event. The resulting B3D+ uis4::etramp8 and B3D+ uis4::etramp10.3 plasmids contained the 3’ UTR of GAPDH following the PfETRAMP ORF.
For replacement of the 3’ UTR of GAPDH with the 3’ UTR of UIS3, a 750 bp fragment of the 3’ UTR of UIS3 was amplified from P. berghei WT ANKA-GFP gDNA by PCR using primers 3’UTR PbUIS3fd-BamHI and 3’UTRPbUIS3 rv-EcoRI. After restriction digestion with BamHI and EcoRI, the 3’ UTR of UIS3 was cloned into the B3D+ uis4::etramp8 and B3D+ uis4::etramp10.3 vectors, generating the B3D uis4::etramp8 and B3D uis4::etramp10.3 vectors. The correct replacements were checked by sequencing. The two vectors were linearized with SacII and KpnI.
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 (gfp-mu3)
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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 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.
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Other details transgene
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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
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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 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
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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