SummaryRMgm-4944
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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) | Not published (yet) |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | RMgm-4935 |
Other information parent line | In this mutant (3194cl1; RMgm-4935; PyXNL-CS GIMO) the endogenous P. yoelii csp gene has been deleted by replacing the csp gene with the drug-selectable marker cassette hdhfr-yfcu. It also expresses GFP and luciferase under the constitutive eef1a promoter. |
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The mutant parasite was generated by | |
Name PI/Researcher | Franke-Fayard B, Janse C.J |
Name Group/Department | Leiden Malaria Group, Department of Parasitology |
Name Institute | Leiden University Medical Center, LUMC |
City | Leiden |
Country | The Netherlands |
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Name of the mutant parasite | |
RMgm number | RMgm-4944 |
Principal name | 3226 (after mosquito passage) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | No |
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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 | PY17X_0405400 | ||||||||||||||||||||||||||
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. yoelii csp gene replaced by P. falciparum csp | ||||||||||||||||||||||||||
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 chimeric parasites where the P. yoelii csp coding sequence (CDS; PY17X_0405400) has been replaced by the P. falciparum csp CDS (Pfcsp; PF3D7_0304600), we used a 2-step GIMO transfection protocol. In the first step we deleted the Pycsp coding open reading frame (ORF) and replaced it with the positive-negative selectable marker, to create a P. yoelii csp deletion GIMO line (PyXNL-CS GIMO). In order to do this, we generated the pL2329 construct that is based on the standard GIMO DNA construct pL1980. This construct contains the positive-negative (hdhfr::yfcu) selection marker (SM) cassette and was used to insert both the Pycsp 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminator sequences respectively. First we replaced the eef1α of the hdhfr::yfcu selectable marker (SM) cassette of the standard GIMO DNA construct pL0034 (MRA-849, www.beiresources.org) with the PCR amplified P. yoelii hsp70 promoter to create pL2137. Next we replaced the eef1α-hdhfr::yfcu selectable marker (SM) cassette from pL1980 with the hsp70-hdhfr::yfcu cassette from pL2137 by digestion of the plasmids with PstI and AgeI. Finally, the Pycsp 5’ and 3’ gene targeting regions were PCR amplified and ligated using KpnI/EcoRI and HindIII/PstI restriction sites to generate pL2329. The construct was linearized using SacII and ScaI restriction sites outside of the 5’ and 3’ TRs before transfection. The construct pL2329 was used to transfect Py-GFP-Luceef1α (1971cl1) parasites using standard methods of 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 csp gene of Py-GFP-Luceef1α parasites is replaced by the hdhfr::yfcu SM cassette, resulting in line 3194. Transfected parasites of line 3194 were cloned by limiting dilution, resulting in the PyXNL-CS GIMO line (line 3194cl1). In the second step we replaced the positive-negative SM in the PyXNL-CS GIMO genome with a Pfcsp gene by GIMO transfection, using construct pL2310. To generate pL2310, we exchanged the Pbcsp 5’ and 3’ UTR regions of pL1972 by the Pycsp 5’ and 3’ UTR regions (using HindIII/BamHI and EcoRI/NotI). The construct was linearized using SacII and ScaI restriction sites before transfection and used to transfect parasites of the PyXNL-CS GIMO line 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 transgenic parasites where the hdhfr::yfcu SM in the csp locus of the PyXNL-CS GIMO line is replaced by the Pfcsp gene, resulting in line 3226. This line has been passed through mosquitoes to collect only the complemented parasites. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP-Luciferase | ||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. The mutant has been generated in the reference line GIMOPy17x (RMgm-688). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p 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_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 | PY17X_0306600 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | P230p; 230 | ||||||||||||||||||
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