RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1317
Malaria parasiteP. berghei
Genotype
Transgene
Transgene Plasmodium: Gene model: PF3D7_1335900; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2)
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)
Phenotype Sporozoite; Liver stage;
Last modified: 17 July 2015, 10:53
  *RMgm-1317
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) : 26139288
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
The mutant parasite was generated by
Name PI/ResearcherLongley RJ, Salman AM, HillAV, Janse CJ, Khan SM
Name Group/DepartmentThe Jenner Institute
Name InstituteUniversity of Oxford
CityOxford
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1317
Principal name2281cl1
Alternative namePfTRAP@PbUIS4
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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
SporozoiteTransgene expression in sporozoites
Liver stageSee additional remarks phenotype
Additional remarks phenotype

Mutant/mutation
The mutant expresses P. falciparum TRAP inder control of the sporozoite/liver stage promoter uis4 and expresses the reporter fusion protein GFP-Luciferase under control of the constitutive eef1a promoter

The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of a GIMO mother line that contains the hdhfr::yfcu selectable marker into the silent 230p locus.

The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). This GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice

Protein (function)

Phenotype
In this study 10 transgenic P. berghei parasites were generated that express P. falciparum proteins that are expressed in the sporozoite/liver stage of P. falciparum.
These transgenic P. berghei parasites expressing the P. falciparum proteins during their sporozoite/liver stage development have been used in a challenge model to test the protective efficacy of novel P. falciparum vaccine candidate antigens. Mice were immunized with the P. falciparum vaccine candidates, followed by  challenge with the transgenic P. berghei sporozoites.

Expression of the P. falciparum antigens in the transgenic parasites was analysed by immunofluorescence assay. Transgenic sporozoites were loaded onto glass slides and fixed with 4% paraformaldehyde. The slides were then blocked with 10% FCS and 1% BSA in PBS before the addition of either monoclonal antibodies to P. berghei CSP (3D11) or P. falciparum CSP (2A10) (MR4, USA), or serum from vaccinated mice. Bound IgG was detected with goat anti-mouse IgG-Alexa Fluor 488 and nuclear DNA stained with 2% Hoechst-33342. Slides were mounted with Fluorescence Mounting Medium and viewed under a Leica DMI-300B microscope.
All transgenic parasites were deemed comparable to wild-type P. berghei in terms of  infectivity to BALB/c mice, with two exceptions: the PfUIS3 transgenic was slightly more potent than wild-type (median 5.3 days time to 1% parasitaemia compared to 6.0 days) (p < 0.0001, Mann-Whitney test), and the PfLSA3 transgenic was slightly less potent (median 6.9 days time to 1% parasitaemia compared to 6.0 days) (p = 0.02, Mann-Whitney test). All transgenic parasites were comparable to wild-type P. berghei in CD-1 mice, with two exceptions: the PfCSP and PfLSA3 transgenics were slightly less potent than wild-type infection (median time to 1% parasitaemia of 6.35 days in wild-type, compared to 6.77 days for the PfCSP transgenic and 6.96 days for PfLSA3).

Additional information


Other mutant
See the link for the other 9 transgenic P. berghei lines expressing P. falciparum proteins that were generated in this study


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_1335900
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2
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 modificationChimeric P. berghei parasites containing the P. falciparum gene of interest at the neutral 230p locus were generated following the ‘gene insertion/marker out’ (GIMO) technology as previously described, using the standard GIMO DNA construct pL0043. This construct contains 5′ and 3′ targeting sequences for the 230p locus as well as a multiple-cloning site for integration of transgene-expression cassettes. These constructs integrate by double crossover homologous recombination and replace the positive-negative selectable marker (SM) (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette with the transgene-expression cassette. The expression cassette contained the transgene flanked by the 5′ and 3′ promoter and transcription terminator sequences of P. berghei UIS4, which were amplified from P. berghei ANKA wild-type (WT) genomic DNA. The coding sequence of the various P. falciparum genes were PCR amplified from P. falciparum genomic DNA (primer sequences are available upon request), apart from LSA1 and LSA3. Due to the large size of these open reading frames the coding sequence was amplified from plasmids used in the generation of the vaccine constructs (and hence codon optimized for expression in mammalian cells). In addition, a reporter-cassette containing GFP::luciferase52, driven by the constitutive P. berghei elongation factor 1 alpha (ef1α ) promoter, was also cloned into each transgene construct. The coding sequence and promoter region of all constructs was confirmed by sequencing. Linearized constructs were introduced into the GIMO parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice through addition of 5-fluorocytosine (5-FC) in drinking water, resulting in negative selection of parasites where the SM in the 230p locus was replaced by the expression/reporter-cassette.
Additional remarks selection procedureThis transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
Other details transgene
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
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP-Luciferase
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 modification
Additional remarks selection procedureThis transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
Other details transgene
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
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