RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4571
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PF3D7_1036400; Gene model (P.falciparum): PF3D7_1036400; Gene product: liver stage antigen 1 (LSA1)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: GFP-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)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene Plasmodium: Gene model: PF3D7_0202100; Gene model (P.falciparum): PF3D7_0202100; Gene product: liver stage associated protein 2; Plasmodium exported protein (PHISTc), unknown function (LSAP2)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: up-regulated in infective sporozoites; early transcribed membrane protein (ETRAMP10.3; UIS4)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3)
Replacement locus: Gene model: PBANKA_1206800; Gene product: zinc finger (CCCH type) protein, putative (s1)
PhenotypeNo phenotype has been described
Last modified: 1 January 2019, 15:49
  *RMgm-4571
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, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
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-4570
Other information parent lineThis GIMO line contains a hdfr/yfcu selection marker (SM) cassette in the neutral s1 (PBANKA_1206800) gene locus. In addition, this line expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
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The mutant parasite was generated by
Name PI/ResearcherHalbroth BR, Spencer AJ, Janse CJ, Khan SM, Salman AM
Name Group/DepartmentThe Jenner Institute
Name InstituteUniversity of Oxford
CityOxford
CountryUK
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Name of the mutant parasite
RMgm numberRMgm-4571
Principal name2403cl1
Alternative namePfLSA1+PfLSAP2
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
This (transgenic) mutant line expresses PfLSAP2 (PF3D7_0202100) under the control of the Pbuis4 regulatory sequences. The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection  in  GIMO parent line RMgm-4570. This parent line  expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.

Protein (function)
Both PfLSAP2 and PfLSA1 proteins have been selected based on their expression in sporozoite/liver stages

Phenotype
Normal development throughout the complete life cycle.

Additional information
From the study:
'A single vaccine expressing two antigens could potentially increase both the size and breadth of antigen specific response, while halving vaccine production cost. In this study we investigated combining two liver stage antigens (PfLSA1 and PfLSAP2; PfTRAP and PfLSA1;  PfTRAP and PfLSAP2), and investigated induction of protective efficacy by co-administration of single-antigen vectors or vaccination with dual-antigen vectors, using simian Adenovirus and Modified Vaccinia Ankara Virus vectors. Efficacy of these vaccines was assessed in mouse malaria challenge models using chimeric P. berghei parasites expressing the relevant Pf antigens.'

Other mutant
 

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_1036400
Gene Model P. falciparum ortholog PF3D7_1036400
Gene productliver stage antigen 1
Gene product: Alternative nameLSA1
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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 markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationWe replaced the positive-negative SM in the genome of the PbANKA-PfPfLSA1+PbΔs1 line (line 2374 cl1) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSA1 and PfLSAP2. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSAP2 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection.

The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4570. This parent line expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameETRAMP10.3
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_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameETRAMP10.3
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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  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-Luciferase
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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 markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationWe replaced the positive-negative SM in the genome of the PbANKA-PfPfLSA1+PbΔs1 line (line 2374 cl1) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSA1 and PfLSAP2. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSAP2 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection.

The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4570. This parent line expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
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 / 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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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_0202100
Gene Model P. falciparum ortholog PF3D7_0202100
Gene productliver stage associated protein 2; Plasmodium exported protein (PHISTc), unknown function
Gene product: Alternative nameLSAP2
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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 markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationWe replaced the positive-negative SM in the genome of the PbANKA-PfPfLSA1+PbΔs1 line (line 2374 cl1) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSA1 and PfLSAP2. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSAP2 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection.

The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4570. This parent line expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
Additional remarks selection procedureThe PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4570. This parent line expresses the PfLSA1 (accession no. PF3D7_1036400) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfLSA1 and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productup-regulated in infective sporozoites; early transcribed membrane protein
Gene product: Alternative nameETRAMP10.3; UIS4
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_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameETRAMP10.3
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_1206800
Gene productzinc finger (CCCH type) protein, putative
Gene product: Alternative names1
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