RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4137
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The P. berghei cs gene replaced by P. vivax cs VK247 (PVP01_0835600)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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: 10 October 2018, 16:45
  *RMgm-4137
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28417968
Reference 2 (PMID number) : 30092798
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherSalman AM, Khan SM, Janse CJ, Reyes-Sandoval A
Name Group/DepartmentThe Jenner Institute, Nuffield Department of Medicinep
Name InstituteUniversity of Oxford, The Henry Wellcome Building for Molecular Physiology
CityOxford
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4137
Principal name2199cl1
Alternative namePbANKA-PvCSP VK247(r)PbCS
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
SporozoiteReduced sporozoite formation (30%)
Liver stageSporozoites showed wild type infectivity to mice after intravenous injection of sporozoites
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei cs gene has been replaced by the P. vivax VK247 cs gene (PVP01_0835600). This has been performed by the GIMO method of transfection. The P. vivax cs gene is under control of the 5'- and 3'-UTR regions of the P. berghei cs gene. The mutant does not contain a drug-selectable marker. The mutant also expresses the fusion protein GFP-Luciferase under control of the constitutive eefia promoter.
(PvCSP insert consisted of Pvcsp-vk247 (GenBank accession number M69059.1; Papua New Guinea strain).

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
Reduced sporozoite formation (30%). Sporozoites showed wild type infectivity to mice after intravenous injection of sporozoites.
See also PMID 30092798 for analyses of mosquito/liver phenotype

Additional information
This chimeric parasite line has been used for analysing (protective) immune responses in mice immunized with different vaccines targeting P. vivax CS. Immunized mice were challenged with chimeric sporozoites expressing P. vivax CS.

Other mutants
In this study two mutants were generated: one expressing the P. vivax CS VK210 protein (RMgm-4136) and the other the P. vivax CS VK247 protein (RMgm-4137).


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Details of the genetic modification
Short description of the mutationThe P. berghei cs gene replaced by P. vivax cs VK247 (PVP01_0835600)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationIn this study two mutants were generated: one expressing the P. vivax CS VK210 protein (see RMgm-4136) and the other the P. vivax CS VK247 protein (see RMgm-4137).
To generate the transgenic parasites where the P. berghei csp gene (PBANKA_0403200) coding sequence (CDS) has been replaced by the CDS of P. vivax csp (PVP01_0835600), we used a 2-step GIMO transfection protocol. In the first step we deleted the P. berghei csp CDS and replaced it with the positive-negative selectable marker, to create a P. berghei csp deletion GIMO line (PbANKA-CSP GIMO). In order to this we generated pL1929 construct that is based on the standard GIMO DNA construct pL003450. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette, and was used to insert both the Pbcsp 5′ and 3′ gene targeting regions (TR), encompassing the full-length promoter and transcription terminators sequences respectively. The linear pL1929 DNA construct was introduced into PbGFP-Luccon parasites using standard methods transfection49. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution51, resulting in the PbANKA-CSP GIMO line (2151cl1). Correct deletion of the P. berghei csp CDS was confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes.
In the second step we replaced the positive-negative SM in the PbANKA-CSP GIMO genome with the CDS of either P. vivax VK210 or VK247 csp by GIMO transfection to create the two P. berghei transgenic CSP replacement lines. This was achieved by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with P. vivax csp CDS sequence. The P. vivax csp CDS was ordered from GeneArt (Regensburg, Germany) (i.e. VK210) or cDNA (VK247) Both the P. vivax VK210 and VK247 CSP constructs (pL1942 and pL1943, respectively) were sequenced to ensure there were no mutations in the P. vivax csp CDS. These constructs were linearized using SacI and PacI restriction enzymes outside of the 5′ and 3′ TRs before transfection. These constructs were used to transfect parasites of the PbANKA-CSP GIMO line (2151cl1) using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of chimeric parasites where the hdhfr::yfcu SM in the csp locus of PbANKA-CSP GIMO line is replaced by the CDS of P. vivax CSP Selected transgenic parasites were cloned by the method of limiting dilution. Correct integration of the constructs into the genome of chimeric parasites was analysed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes. This method creates transgenic ‘gene replacement’ P. berghei parasites that do not contain P. berghei csp gene CDS but express either P. vivax VK210 (PbANKA-PvCSP VK210(r)PbCS; 2196cl1) or VK247 csp (PbANKA-PvCSP VK247(r)PbCS; 2199cl1) under the control of the P. berghei csp regulatory sequences.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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