RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-594
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: firefly luciferase
Promoter: Gene model: PBANKA_1003000; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative | sequestrin | 6-cysteine protein (LISP2)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type and/or d-type unit))
Transgene
 Transgene not Plasmodium: Renilla luciferase
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)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-type and/or d-type unit))
Phenotype Liver stage;
Last modified: 6 November 2013, 18:51
  *RMgm-594
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) : 21048918
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/ResearcherS. Helm; V.T. Heussler
Name Group/DepartmentMalaria Laboratory 1
Name InstituteBernhard Nocht Institute for Tropical Medicine
CityHamburg
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-594
Principal namePbFL103464RLef1a
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 different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageExpression of luciferase under the control of the PBANKA_100300 promoter in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant (PbFL103464RLef1a) expresses firefly luciferase under control of the PBANKA_100300 promoter and Renilla luciferase under control of the constitutive eef1a promoter

Protein (function)
No data is presented on the function of sequestrin. This gene has been identified in a DNA microarray study as expressed solely during the liver stage. Expression experiments presented in this paper confirm the liver stage expression.

Phenotype
This mutant has been used to determine the relative expression level of luciferase under the control of the PBANKA_100300 promoter in blood, mosquito and liver stages.

Additional information
Analysis of GFP expression has its limitations as it does not allow an accurate quantitative analysis of promoter activity. It was therefore decided to clone the 989bp PBANKA_100300 promoter region in front of the firefly luciferase (FL) gene and to normalize the measurement of firefly luciferase activity by including in the same plasmid a Renilla luciferase (RL) gene under the control of the constitutive ef1a promoter. This plasmid was named pFL103464RLef1a. As a control, a further plasmid was constructed in which both luciferase genes were under the control of the constitutive promoter (pFLef1aRLef1a). Both plasmids were transfected separately into P. berghei and the parasite strains PbFL103464RLef1a and PbFLef1aRLef1a (not included in the RMgm database),were established.
The plasmids used allow integration into the P. berghei d/c ssu rrna locus, but are also able to persist in the parasite population as episomes. As both luciferases are encoded on each plasmid and therefore the genes are always present in equal numbers, it was not necessary to analyze genetically whether the parasite populations contained integrated or episomal gene copies or (as is most likely) a mixture of both.

Other mutants
RMgm-593: A mutant expressing gfp under control of the PBANKA_100300 promoter
RMgm-595: A mutant expressing firefly luciferase under control of the PBANKA_040200 promoter and Renilla luciferase under control of the constitutive eef1a promoter

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namefirefly luciferase
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ApaI, SacII
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAnalysis of GFP expression has its limitations as it does not allow an accurate quantitative analysis of promoter activity. It was therefore decided to clone the 989bp PBANKA_100300 promoter region in front of the firefly luciferase (FL) gene and to normalize the measurement of firefly luciferase activity by including in the same plasmid a Renilla luciferase (RL) gene under the control of a constitutive promoter. This plasmid was named pFL103464RLef1a. As a control, a further plasmid was constructed in which both luciferase genes were under the control of the constitutive promoter (pFLef1aRLef1a). Both plasmids were transfected separately into P. berghei and the parasite strains PbFL103464RLef1a and PbFLef1aRLef1a (not included in the RMgm database),were established.
The plasmids used allow integration into the P. berghei d/c ssu rRNA locus, but are also able to persist in the parasite population as episomes. As both luciferases are encoded on each plasmid and therefore the genes are always present in equal numbers, it was not necessary to analyze genetically whether the parasite populations contained integrated or episomal gene copies or (as is most likely) a mixture of both.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1003000
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative | sequestrin | 6-cysteine protein
Gene product: Alternative nameLISP2
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type and/or d-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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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameRenilla luciferase
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid ApaI, SacII
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAnalysis of GFP expression has its limitations as it does not allow an accurate quantitative analysis of promoter activity. It was therefore decided to clone the 989bp PBANKA_100300 promoter region in front of the firefly luciferase (FL) gene and to normalize the measurement of firefly luciferase activity by including in the same plasmid a Renilla luciferase (RL) gene under the control of a constitutive promoter. This plasmid was named pFL103464RLef1a. As a control, a further plasmid was constructed in which both luciferase genes were under the control of the constitutive promoter (pFLef1aRLef1a). Both plasmids were transfected separately into P. berghei and the parasite strains PbFL103464RLef1a and PbFLef1aRLef1a (not included in the RMgm database),were established.
The plasmids used allow integration into the P. berghei d/c ssu rRNA locus, but are also able to persist in the parasite population as episomes. As both luciferases are encoded on each plasmid and therefore the genes are always present in equal numbers, it was not necessary to analyze genetically whether the parasite populations contained integrated or episomal gene copies or (as is most likely) a mixture of both.
Additional remarks selection procedure
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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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-type and/or d-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