SummaryRMgm-4112
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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) : 30469323 |
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-4112 |
Principal name | 2632cl1 |
Alternative name | PbANKA-PfTRAP (r)PbTRAP |
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_1349800 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||||||||||
Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||||||||||
Gene product: Alternative name | TRAP; SSP2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The P. berghei trap gene replaced by P. falciparum trap | ||||||||||||||||||||||||||
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 trap coding sequence (CDS; Pbtrap; PBANKA_134980) has been replaced by the P. falciparum trap CDS (Pftrap; PF3D7_1335900), we used a 2-step GIMO transfection protocol. In the first step we deleted the Pbtrap CDS and replaced it with the positive-negative selectable marker, to create a P. berghei trap deletion GIMO line (PbANKA-TRAP GIMO). In order to this we generated pL2111 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 Pbtrap 5’ and 3’ gene targeting regions (TR), encompassing the full length promoter and transcription terminators sequences respectively. The linear pL2111 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-TRAP GIMO line (line 2564). Correct deletion of the Pbtrap CDS was confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes as described. Primers used for PCR genotyping are listed in Table S1 and S2. In the second step we replaced the positive-negative SM in the PbANKA-TRAP GIMO genome with the Pftrap CDS by GIMO transfection to create the P. berghei chimeric TRAP replacement line. This was achieved by modifying the construct used in the first step (pL2111); specifically, the hdfhr::yfcu SM cassette was removed and replaced with Pftrap CDS sequence, generating plasmid pL2127. The Pftrap CDS was amplified from the NF54 strain of P. falciparum (Pftrap; PF3D7_1335900). The pL2127 construct was sequenced to ensure there were no mutations in the Pftrap CDS. The construct was linearized using ApaI and KasI restriction enzymes outside of the 5’ and 3’ TRs before transfection. The construct was used to transfect parasites of the PbANKA-TRAP GIMO line (line 2564cl3) 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 trap locus of PbANKA-TRAP GIMO line is replaced by the CDS of Pftrap. Selected chimeric 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 chimeric ‘gene replacement’ P. berghei parasites that lack the Pbtrap CDS but express PfTRAP (PbANKA-PfTRAP (r)PbTRAP; line 2632cl1) under the control of the Pbtrap 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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