RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1228
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0800500; Gene model (P.falciparum): PF3D7_1252200; Gene product: chitinase (CHT1)
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)
PhenotypeNo phenotype has been described
Last modified: 25 March 2015, 18:06
  *RMgm-1228
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) : 25728487
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/ResearcherUkegbu CV; Vlachou D
Name Group/DepartmentDepartment of Life Sciences
Name InstituteImperial College London
CityLondon
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-1228
Principal namewt_cht(p)red(230p)
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 stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses mCherry under the control of the  promoter of the chitinase gene (cht1; PBANKA_080050) . The transgene expression cassette is introduced into the silent p230p locus.

Protein (function)

Phenotype
In the mutant transcription of the transgene began 2 hours post fertilization and the fluorescent reporter was detected in developing zygotes/ookinets at 12 hours post fertilization

Growth and multiplication of blood, mosquito and liver stages is comparable to that of wild type parasites

Additional information

Other mutants

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor the generation of the wt_cht(p)red230p and wt_cht(p)green230p lines, the pcht(p)mCherry and pcht(p)gfp vectors respectively were constructed. Firstly, a 820 bp fragment directly upstream of the cht1 open reading frame (ORF) was amplified from P. berghei genomic DNA as an AflII/BamHI fragment and cloned into the pCR2.1-TOPO vector (Life technologies). To construct the pcht(p)mCherry vector, the plasmid pmCherrycon (see below)) served as a backbone for this vector. The ef1αp promoter of pmCherrycon was replaced by the AflII/BamHI fragment of the chtp promoter. Similarly, to construct the pcht(p)gfp vector, the plasmid pcht(p)mCherry served as a backbone for this vector. The BamHI fragment of the mCherry gene of pcht(p)mCherry plasmid was replaced with the BamHI fragment of pL0018 (see below)containing the gfp mutant 3 gene.

To introduce a constitutively expressed mCherry cassette into the P. berghei ANKA 15cy1A genome and generate the wt_red230p (Rmgm-1227) stable transgenic line, the pmCherrycon vector plasmid was constructed. The plasmid pL0018 (MRA-787, MR4) served as a backbone for this vector. The plasmid pL0018 contains two expression cassettes, one for the expression of the selectable marker Toxoplasma gondii dhfr (tgdhfr) under the control of the P. berghei dhfr promoter (dhfrp) and a second under the control of the P. berghei ef1αp that allows constitutive expression of any inserted downstream transgene. The 230p cassette allows integration via double crossover homologous recombination. mCherry was amplified from pmCherry vector (Clonetech) as a BamHI fragment and cloned into the pCR2.1-TOPO vector (Invitrogen). The BamHI fragment of the GFP mutant 3 gene of plasmid pL0018 was replaced with the BamHI mCherry gene fragment.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0800500
Gene Model P. falciparum ortholog PF3D7_1252200
Gene productchitinase
Gene product: Alternative nameCHT1
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