SummaryRMgm-5235
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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) : 34504134 |
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 | RMgm-2151 |
Other information parent line | The mutant lacks expression of CSP. In the mutant the endogenous P. berghei csp gene has 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 | Giminez, AM, Reyes-Sandoval A |
Name Group/Department | Instituto Politécnico Nacional, IPN |
Name Institute | Instituto Politécnico Nacional, IPN |
City | Mexico City |
Country | Mexico |
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Name of the mutant parasite | |
RMgm number | RMgm-5235 |
Principal name | 2710cl1,2 |
Alternative name | |
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 | Wild-type numbers of mature oocysts are formed. However, no visible spz inside oocysts, and only very few salivary gland spz were observed. |
Sporozoite | no visible spz inside oocysts, and only very few salivary gland spz were observed. |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) |
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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 | CS; CSP | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | P. berghei csp replaced with the 'P. vivax like' csp (locus PVU09738). | ||||||||||||||||||||||||||
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 P. berghei replacement line, we replaced the P. berghei csp coding sequence (CDS; Pbcsp; PBANKA_0403200) with the PvCSP-P. vivax-like CDS (Locus PVU09738, Accession U09738) using a 2-step GIMO transfection protocol. In the first step, we deleted the P. berghei csp CDS and replaced it with a positive–negative selectable marker to create a P. berghei csp deletion GIMO line (PbANKA-CSP GIMO). The construct (pL1929) used and the generation of the PbANKA-CSP GIMO line (line 2251cl1). This construct contains the positive–negative (hdhfr::yfcu) SM cassette and was used to insert both the Pbcsp 5′ and 3′ gene targeting regions (TRs), encompassing the full-length promoter and transcription terminator sequences, respectively, and was transfected into PbGFP-Luccon parasites (676m1cl1) using standard transfection methods. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water. Transfected parasites were cloned using the limiting dilution method, resulting in the PbANKA-CSP GIMO line (line 2251 cl1). In the second step, we replaced the positive–negative SM in the PbANKA-CSP GIMO genome with the PvCSP-P. vivax-like CDS by GIMO transfection to create the P. berghei chimeric Pb-PvG10 replacement line Pb-PvG10(r). This line was obtained by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PvCSP-P. vivax-like CDS, generating plasmid pL2161. The PvCSP-P. vivax-like CDS was ordered from GeneArt Gene Synthesis—Thermo Fisher Scientific. The pL2161 construct was sequenced to ensure that no mutations were present in the PvCSP-P. vivax-like CDS during the cloning process. The construct was linearized using AflII and SacI restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the PbANKA-CSP GIMO line (line 2251 cl1) using standard methods of GIMO transfection to generate a single replacement gene chimeric parasite. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water. Negative selection results in the selection of chimeric parasites where the hdhfr::yfcu SM in the csp locus on chromosome 4 of the PbANKA-CSP GIMO line is replaced with the PvCSP-P. vivax-like CDS. Selected chimeric parasites were cloned using the limiting dilution method. | ||||||||||||||||||||||||||
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 | The GFP-Luciferase gene (1 copy) has been inserted into the 230p locus by double cross-over integration. | ||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence. | ||||||||||||||||||
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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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