RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5201
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0508300; Gene model (P.falciparum): PF3D7_1024100; Gene product: conserved Plasmodium protein, unknown function (OMG1, ookinete maturation gene 1)
Name tag: mNeonGreen fluorescent protein (mNG)
Transgene
 Transgene not Plasmodium: Cas9 from Streptococcus pyogenes
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type unit))
Phenotype Fertilization and ookinete;
Last modified: 11 August 2022, 12:05
  *RMgm-5201
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) Reference 1 (PMID number) : 35947628
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 RMgm-4870
Other information parent lineThe mutant (RMgm-4870) contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double-stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which expresses it constitutively. It does not contain a drug-selectable marker that has been removed by negative selection
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The mutant parasite was generated by
Name PI/ResearcherNishi T, Yuda M
Name Group/DepartmentLaboratory of Medical Zoology, Department of Medicine
Name InstituteMie University
CityMie, Tsu
CountryJapan
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Name of the mutant parasite
RMgm numberRMgm-5201
Principal nameOMG1::mNG
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 ookineteTranscripts of omg1 were significantly upregulated at 6 h after activation of gametocytes (hoc) compared to non-activated gametocytes, with a log2(fold change) of 3.33, suggesting its transcription after fertilization. In the blood stage, both female and male gametocytes of OMG1::mNG showed no fluorescent signal. After fertilization, a weak signal started to appear at 6 hoc. This signal intensified in retort-form ookinetes and was still observed in matured ookinetes. Fluorescence was observed along the periphery of ookinetes but was absent in the zygote remnant and the apical end.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mNeonGreen fluorescent protein (mNG)-tagged version of OMG1.

In addition, the mutant contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively. It does not contain a drug-selectable marker which has been removed by negative selection.

The mutant does not contain a drug selectable marker

Protein (function)
The OMG1 protein was identified as a target of AP2-Z (an AP2 transcription factor expressed in zygotes); see also RMgm-5200).
Although the target genes of AP2-Z largely overlapped with those of AP2-O, they also contained a considerable number of unique targets, most of which have not yet been functionally assessed. One of these unique target genes with an unknown function, was OMG1 (PBANKA_0508300)

Phenotype
Transcripts of omg1 were significantly upregulated at 6 h after activation of gametocytes (hoc) compared to non-activated gametocytes, with a log2(fold change) of 3.33, suggesting its transcription after fertilization. By fusing mNG (mNeonGreen fluorescent protein (mNG)) to omg1 using CRISPR/Cas9 (OMG1::mNG) we investigated the omg1 expression pattern during ookinete development. In the blood stage, both female and male gametocytes of OMG1::mNG showed no fluorescent signal. After fertilization, a weak signal started to appear at 6 hoc. This signal intensified in retort-form ookinetes and was still observed in matured ookinetes. Fluorescence was observed along the periphery of ookinetes but was absent in the zygote remnant and the apical end. This pattern of distribution is similar to that of several known IMC components, such as IMC1h and IMC1iNo GFP expression in female gametocytes. .

See also mutant RMgm-5202 that lacks expression of OMG1, showing delayed ookinete maturation and reduced oocyst production 

Additional information

Other mutants
  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0508300
Gene Model P. falciparum ortholog PF3D7_1024100
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameOMG1, ookinete maturation gene 1
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Details of the genetic modification
Name of the tagmNeonGreen fluorescent protein (mNG)
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 parasiteunknown
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe previously reported CRISPR/Cas9 system was used to generate transgenic parasites by CRISPR/Cas9 (see RMgm-4870). Briefly, PbCas9 parasites, which constitutively express Cas9 endonuclease, were transfected with donor DNA prepared by the overlap PCR method and sgRNA vector by electroporation. After transfection, mice infected with the transfectants were treated with 70 μg/mL of pyrimethamine (Sigma) in their drinking water for three days to select the desired mutants. All clonal parasites were obtained by limiting dilution methods, and genotyping was performed by PCR and Sanger sequencing.
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 nameCas9 from Streptococcus pyogenes
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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 parasiteNo selectable marker
Promoter of the selectable markerN.A.
Selection (positive) procedureN.A.
Selection (negative) procedureN.A.
Additional remarks genetic modificationCas9 from Streptococcus pyogenes was used in this study. This Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively.To introduce the expression cassette of Cas9 nuclease in the genome of P. berghei, the pcssu-Cas9-hy plasmid was constructed. The pcssu-Cas9-hy contained not only the Cas9 expression cassette but also the hdhfr–yfcu expression cassette as the positive/negative selection marker. The human dihydrofolate reductase gene, i.e., hdhfr confers pyrimethamine resistance to the parasites and was used as a positive selectable marker. The yfcu gene is a fusion gene of yeast cytosine deaminase and uridyl-phosphoribosyltransferase. The parasite that expresses the yfcu gene is killed by 5-FC, and the yfcu can thus be used as a negative selection marker. The Cas9 cassette and the hdhfr–yfcu cassette contained the 3′UTR of hsp70, which was used for the termination of transcription of both genes. To generate the hdhfr–yfcu fusion gene cassette, hdhfr with the ef1α promoter was amplified by PCR using pSK-125 as a template and the primers Pef1α-F and hdhfr-R, while yfcu with the 3′UTR of hsp70 was amplified from pfgRNA15 with the primers yfcu-F and hsp3UTR-R. These two amplified fragments were fused by PCR. In the fused fragment, the termination codon of hdhfr was eliminated to generate the fusion gene. The resulting hdhfr–yfcu cassette was cloned into the SalI/BamHI sites of pSK-1, resulting in the pSK-1-hy plasmid. The Cas9 expression cassette was excised by NheI/SalI from the pfCas9 plasmid15 and cloned into the pSK-1-hy plasmid, resulting in the pSK-1-Cas9-hy plasmid. To integrate the Cas9 and hdhf-yfcu cassettes into the genomic locus of the rRNA Ctype subunit (cssu) on chromosome 5, two partial sequences, HDR1 and 2, of the cssu were amplified and cloned into the KpnI/XhoI site and the BamHI/NotI site of pSK-1-Cas9-hy, resulting in the pcssu-Cas9-hy plasmid.
Additional remarks selection procedureThe mutant does not contain a drug-selectable marker (hdhfr-yfcu) which has been removed by negative selection
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Other details transgene
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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
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3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 namesmall subunit ribosomal rna gene (c-type unit)
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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren