Back to search resultsSummaryRMgm-4111
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Successful modification | The 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) : 28179403 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | P. berghei ANKA 676m1cl1 (RMgm-29) |
Other information parent line | 676m1cl1 (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). |
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The mutant parasite was generated by | |
Name PI/Researcher | Salman AM, Khan SM, Janse CJ, |
Name Group/Department | Leiden Malaria Research Group |
Name Institute | Leiden University Medical Center |
City | Leiden |
Country | The Netherlands |
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Name of the mutant parasite | |
RMgm number | RMgm-4111 |
Principal name | 2321cl3 |
Alternative name | PbANKA-PvCelTOS(r)PbCelTOS |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1432300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1216600 | ||||||||||||||||||||||||||
Gene product | cell traversal protein for ookinetes and sporozoites | ||||||||||||||||||||||||||
Gene product: Alternative name | CelTOS | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The P. berghei celtos gene replaced by P. vivax (Sal I) celtos (PVX_123510) | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate the chimeric parasites where the P. berghei celtos coding sequence (CDS; Pbceltos; PBANKA_1432300) has been replaced by the P. vivax celtos CDS (Pvceltos; PVX_123510), 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 Pvceltos 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 Pvceltos CDS sequence, generating plasmid pL2017. TThe Pvceltos CDS was amplified from DNA of P. vivax Sal I strain (Pvceltos; PVX_123510). The pL2017 construct was sequenced to ensure there were no mutations in the Pvceltos 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 Pvceltos (line 2320). Selected chimeric parasites were cloned by 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 chimeric ‘gene replacement’ P. berghei parasites that lack the Pbceltos CDS but express PvCelTOS (PbANKA-PvCelTOS(r)PbCelTOS; line 2320cl2) under the control of the Pbceltos regulatory sequences. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||
Selectable marker used to select the mutant parasite | gfp (FACS) | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
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 product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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