RMgmDB - Rodent Malaria genetically modified Parasites

Back to search results

Summary

RMgm-4066
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1432300; Gene model (P.falciparum): PF3D7_1216600; Gene product: cell traversal protein for ookinetes and sporozoites (CelTOS)
Details mutation: The P. berghei celtos gene replaced by P. falciparum celtos
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)
PhenotypeNo phenotype has been described
Last modified: 23 January 2017, 12:49
  *RMgm-4066
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) : 27895131
MR4 number
top of page
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).
top of page
The mutant parasite was generated by
Name PI/ResearcherEspinosa DA, Zavala F, Gutierrez GM, Salman AM, Khan SM, Janse CJ,
Name Group/DepartmentDept. of Molecular Microbiology and Immunology, Malaria Research Institute
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
top of page
Name of the mutant parasite
RMgm numberRMgm-4066
Principal name2258cl2
Alternative namePbANKA-PfCelTOS(r)PbCelTOS
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
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
In the mutant the endogenous P. berghei celtos gene has been replaced by the P. falciparum celtos gene. This has been performed by the GIMO method of transfection. The P. falciparum celtos gene is under control of the 5'- and 3'-UTR regions of the P. berghei celtos 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.

Protein (function)
CelTOS is localized to the micronemes of ookinetes and (salivary gland) sporozoites and is secreted. It plays a role in migration of ookinetes through mosquito midgut cells and traversal of sporozoites through heptocytes

Phenotype
Normal development throughout the complete life cycle showing complementation of P. berghei CELTOS by P. falciparum CELTOS

Additional information
Using the newly developed rodent malaria chimeric parasite expressing the P. falciparum CELTOS, the protective effect of in vivo immune responses elicited by vaccination was evaluated and  the neutralizing capacity of monoclonal antibodies specific against PfCelTOS was assessed.

Other mutants

  Mutated: Mutant parasite with a mutated gene
top of page
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1432300
Gene Model P. falciparum ortholog PF3D7_1216600
Gene productcell traversal protein for ookinetes and sporozoites
Gene product: Alternative nameCelTOS
top of page
Details of the genetic modification
Short description of the mutationThe P. berghei celtos gene replaced by P. falciparum celtos
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 modificationTo generate the chimeric parasites where the P. berghei celtos coding sequence (CDS; Pbceltos; PBANKA_1432300) has been replaced by the P. falciparum celtos CDS (Pfceltos; PF3D7_1216600), we used a 2-step gene insertion/marker out transfection protocol (GIMO). In the first step we deleted the Pbceltos CDS and replaced it with the positive-negative selectable marker, to create a P. berghei celtos deletion GIMO line (PbANKA-CelTOS GIMO). In order to do this, we generated the pL1960 construct that is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette, and was used to insert both the Pbceltos 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. The linear pL1960 DNA construct was introduced into PbGFP-Luccon parasites (676m1cl1) using standard methods of transfection. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution, resulting in the PbANKA-CelTOS GIMO line (line 2217).

In the second step we replaced the positive-negative SM in the PbANKA-CelTOS GIMO genome with the Pfceltos CDS by GIMO transfection to create the P. berghei chimeric CelTOS replacement line. This was achieved by modifying the construct used in the first step (pL1960); specifically, the hdfhr::yfcu SM cassette was removed and replaced with Pfceltos CDS sequence, generating plasmid pL1971. The Pfceltos CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL1971 construct was sequenced to ensure there were no mutations in the Pfceltos CDS. The construct was linearized using ApaI and NotI restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the PbANKA-CELTOS GIMO line (line 2217cl1) 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 celtos locus of PbANKA-CelTOS GIMO line is replaced by the CDS of Pfceltos (line 2258). Selected chimeric parasites were cloned by limiting dilution
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
top of page
  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
top of page
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
top of page
Other details transgene
top of page
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
top of page
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
top of page
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