Back to search resultsSummaryRMgm-4137
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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) : 28417968 Reference 2 (PMID number) : 30092798 |
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, Reyes-Sandoval A |
Name Group/Department | The Jenner Institute, Nuffield Department of Medicinep |
Name Institute | University of Oxford, The Henry Wellcome Building for Molecular Physiology |
City | Oxford |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-4137 |
Principal name | 2199cl1 |
Alternative name | PbANKA-PvCSP VK247(r)PbCS |
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 | Reduced sporozoite formation (30%) |
Liver stage | Sporozoites showed wild type infectivity to mice after intravenous injection of sporozoites |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0403200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0304600 | ||||||||||||||||||||||||||
Gene product | circumsporozoite (CS) protein | ||||||||||||||||||||||||||
Gene product: Alternative name | CSP | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The P. berghei cs gene replaced by P. vivax cs VK247 (PVP01_0835600) | ||||||||||||||||||||||||||
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 | In 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 | |||||||||||||||||||||||||||
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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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