RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4916
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene Plasmodium: Gene model: PF3D7_0317100; Gene model (P.falciparum): PF3D7_0317100; Gene product: 6-cysteine protein (B9)
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_1206800; Gene product: zinc finger (CCCH type) protein, putative (S1)
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, 11:13
  *RMgm-4916
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 RMgm-4434
Other information parent lineGIMO(S1)-PbANKA (RMgm-4434, 2149cl2) 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 silent s1 locus (PBANKA_1206800) 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 into the s1 locus. It also expresses the fusion gene GFP-Luciferase under the constitutive eef1a promoter (introduced into the silent 230p locus)
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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-4916
Principal name2392cl2
Alternative namePbB9(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 B9. The P. falciparum gene is introduced as an additional copy into the silent s1 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 s1 locus.

The mutant has been generated in the reference line GIMO(S1)-PbANKA (RMgm-4434, 2149cl2) 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 silent s1 locus (PBANKA_1206800) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter.  It also expresses the fusion gene GFP-Luciferase under the constitutive eef1a promoter (introduced into the silent 230p locus) 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_0317100
Gene Model P. falciparum ortholog PF3D7_0317100
Gene product6-cysteine protein
Gene product: Alternative nameB9
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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 modificationTwo ‘additional copy’ chimeric P. berghei ANKA parasites (expressing B9 and MAEBL were generated by introducing the P. falciparum gene expression cassette into a neutral Pbs1 gene (PBANKA_1206800) of 676m1cl1 parasites using the 2-step ‘gene insertion/marker out’ (GIMO) transfection protocol. In the first step, a linear DNA construct (plasmid pL1928) that has a positive/negative hdhfr::yfcu SM cassette was introduced into the Pbs1 gene locus to obtain Pbs1 GIMO mother line 2149cl2 (RMgm-4434, www.pberghei.eu). Plasmid pL1928 was generated using the GIMO-plasmid pL0034 (MRA-849, www.beiresources.org), containing the hdhfr::yfcu SM. This plasmid was used to insert both the 5’ (0.7 Kb) and 3’ gene (0.8 Kb) targeting regions (TR) of Pbs1. The Pbs1 5’utr was PCR amplified using primer pair 1003/1004 and ligated into pL0034 using ApaI and KpnI restriction sites. Next, the Pbs1 3’utr was PCR amplified using the primer pair 1005/1006 and ligated using KasI and NotI restriction sites, resulting in plasmid pL1928 (primers described in Table S1 and S3). Plasmid pL1928 was linearized using KasI and ApaI restriction sites and transfected into 676m1cl1 parasites using GIMO-transfection. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Positive selection results in selection of transgenic parasites where the s1 gene is replaced by the hdhfr::yfcu SM, resulting in line 2149. Parasites of line 2149 were cloned by limiting dilution and clone 2149cl2 (Pbs1 GIMO) line was used for step 2. Correct integration of SM cassette into the Pbs1 locus was confirmed by diagnostic PCR analysis of genomic DNA (gDNA) and Southern analysis of pulsed-field gel electrophoresis-separated chromosomes. In step 2, we replaced the hdhfr::yfcu SM in Pbs1 GIMO parasites with the transgene expression cassette. This was achieved by first generating the plasmid, pL2046, by ligating the 5’utr and 3’utr sequences of Pbuis4 from pL2281 into pL1928 using restriction sites KpnI and PstI to obtain pL2046. The ORFs of B9 and MAEBL were amplified from P. falciparum (NF54 strain) genomic DNA using primers described in Table S1 and S3 and inserted into plasmid pL2046 in between the 5’utr and 3’utr of Pbuis4 using the restriction sites NotI and BsaBI to obtain pL2041 and pL2289, respectively. Plasmid pL2041 was linearized using ApaI and KasI and pL2289 with HindIII and transfected in Pbs1-GIMO parasites using GIMO-transfection. Transfected parasites were selected by applying negative selection 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 s1 locus is replaced by the P. falciparum gene expression cassette
Additional remarks selection procedureThis transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMO(S1)PbANKA (RMgm-4434). The GIMO mother line is used for introduction of transgenes into the modified s1 locus through transfection with constructs that target the s1 locus. These constructs insert into the s1 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_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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  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 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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren