RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4914
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PF3D7_0408700; Gene model (P.falciparum): PF3D7_0408700; Gene product: perforin-like protein 1 (PLP1, PPLP1, SPECT2)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3; UIS4)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3, UIS4)
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: 13 July 2021, 11:12
  *RMgm-4914
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) : 34252120
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 GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687, 1596cl1) 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).
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The mutant parasite was generated by
Name PI/ResearcherKolli SK, Salman AM, Hill AVS, Janse CJ
Name Group/DepartmentMalaria research group, Department of Parasitology
Name InstituteLeiden University Medical Center, LUMC
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4914
Principal name3039cl1
Alternative namePbPfPLP1/SPECT2(uis4)
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
SporozoiteNormal numbers of sporozoites; P. falciparum transgene expression in sporozoites; sporozoites have reduced infectivity (as shown by reduced or absence of parasite liver loads after intravenous injection of sporozoites and absence of blood-stage infection or prolonged prepatent period)
Liver stageSporozoites have reduced infectivity (as shown by reduced or absence of parasite liver loads after intravenous injection of sporozoites and absence of blood-stage infection or prolonged prepatent period)
Additional remarks phenotype

Mutant/mutation
The mutant expresses P. falciparum PLP1/SPECT2. The P. falciparum gene is introduced as an additional copy into the silent p230p locus under 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
Normal numbers of sporozoites; P. falciparum transgene expression in sporozoites; sporozoites have reduced infectivity (as shown by reduced or absence of parasite liver loads after intravenous injection of sporozoites and absence of blood-stage infection or prolonged prepatent period).

The failure to establish a liver infection like wild type parasites, indicates that expression of the P. falciparum protein in sporozoites (as an additional copy) has a negative impact of on sporozoite infectivity/establishment of a liver infection.

In this study 14 transgenic P. berghei parasites were generated that express P. falciparum proteins that are expressed in the sporozoite/liver stage of P. falciparum.
Six of these chimeric P. berghei parasites that produce infective sporozoites have been used in a challenge model to test the protective efficacy of novel P. falciparum viral vectored 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 sporozoites was analysed by immunofluorescence assay (using serum from vaccinated mice)  

Additional information

Other mutant
See the link for the other 14 transgenic P. berghei lines expressing P. falciparum proteins that were generated in this study

  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_0408700
Gene Model P. falciparum ortholog PF3D7_0408700
Gene productperforin-like protein 1
Gene product: Alternative namePLP1, PPLP1, SPECT2
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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 modification‘Additional copy’ chimeric P. berghei ANKA parasites expressing different P. falciparum proteins were generated by replacing the positive negative drug selectable marker (SM) cassette in the 230p (PBANKA_0306000) locus of 1596cl1 parasites with the transgene expression cassette by ‘gene insertion/marker out’ (GIMO) technology. This was achieved by first generating the basic plasmid, pL2281, based on the basic 230p targeting plasmid pL0043. The Pbuis4 5’utr (1.5 Kb) and 3’utr (1 Kb) sequences were amplified using the primers 7169/7170 and 7171/7172, respectively, and introduced into pL0043 using the restriction sites PstI/NotI and EcoRV/KpnI, respectively, to obtain pL2005. Next, a gfp::luc fusion reporter gene under the constitutive Pbeef1a promoter was amplified from the plasmid pL0027, (MRA-796, https://www.beiresources.org) using the primers 7291/7292, and introduced at the KpnI restriction site of pL0025 to obtain pL2281. The open reading frame (ORF) of the P. falciparum genes were amplified from P. falciparum (NF54 strain) genomic DNA and the ORFs were inserted into plasmid pL2281 in between the 5’utr and 3’utr of Pbuis4 using the restriction sites NotI and BsaBI to obtain the final constructs that contain both the P. falciparum gene expression cassette and the gfp::luc expression cassette. All constructs were linearized with SacII and introduced into 1596cl1 parasites using standard methods of GIMO-transfection. Transfected parasites were selected by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of the mice. Negative selection results in the selection of chimeric parasites where the hdhfr::yfcu SM in the 230p locus is replaced by the P. falciparum gene expression cassette and the gfp::luc expression cassette. All chimeric parasites were cloned by the method of limiting dilution
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.
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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; 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, UIS4
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 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.
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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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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