Back to search resultsSummaryRMgm-5060
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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, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35994647 |
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-5058 |
Other information parent line | This mutant lacks expression of QC and expresses mCherry and luciferase under control of constitutive promoters. In this QC-GIMO line the qc open reading frame is replaced with a hdhfr-yfcu selectable marker cassette. |
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The mutant parasite was generated by | |
Name PI/Researcher | Kolli SK, Janse CJ |
Name Group/Department | Malaria Research 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-5060 |
Principal name | 3276 |
Alternative name | Pbqc(CD) |
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 | Normal numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in >50% of QC-null infected mosquitoes, while such oocysts were absent in WT-infected mosquitoes |
Sporozoite | Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Light-microscopy analysis of oocysts between day 14 and 21 showed the presence of aberrant, enlarged, melanized sporozoites, either still inside or during release from oocysts. On day 21 p.i. non-motile, enlarged sporozoites covered by melanin were found in the hemocoel, often in clusters or attached to salivary glands. In contrast, most QC-null sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization. |
Liver stage | Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Wild type development of liver stages in vitro. |
Additional remarks phenotype | Mutant/mutation Protein (function) A single gene encoding a glutaminyl cyclase (QC), named glutaminyl-peptide cyclotransferase, has been identified by electronic annotation in all sequenced Plasmodium genomes. Plasmodium QCs share 70-76% sequence similarity and 50-54% identity and contain a transmembrane domain. QC of the human malaria parasite P. falciparum (PfQC) shows 21-27% identity to QCs of various 64 bacteria and the plant Carica papaya (CpQC). All 9 amino acids of the catalytic site of bacterial and plant QCs are conserved in Plasmodium QC. Normal numbers of oocysts are produced. On day 10 after infection melanized oocysts are present in >50% of QC-null infected mosquitoes, while such oocysts were absent in WT-infected mosquitoes.
Reduced numbers of salivary gland sporozoites. Salivary gland sporozoites show wild-type morphology, motility and infectivity to hepatocytes in vitro. Light-microscopy analysis of oocysts between day 14 and 21 showed the presence of aberrant, enlarged, melanized sporozoites, either still inside or during release from oocysts. On day 21 p.i. non-motile, enlarged sporozoites covered by melanin were found in the hemocoel, often in clusters or attached to salivary glands. In contrast, most QC-null sporozoites inside salivary glands had a WT-like morphology, i.e. long and slender, without signs of melanization. Wild type development of liver stages in vitro after infection of hepatocytes with salivary gland sporozoites. Additional information From the Abstract: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1310700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1446900 | ||||||||||||||||||||||||||
Gene product | glutaminyl-peptide cyclotransferase, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | glutamyl cyclase, QC | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | two point mutations in the catalytic site of QC (F103A, Q105A) | ||||||||||||||||||||||||||
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 | No selectable marker | ||||||||||||||||||||||||||
Promoter of the selectable marker | No | ||||||||||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate Pbqc(CD) parasites, expressing the catalytic dead enzyme QCCD, DNA construct pL2348 was generated. Partial fragments of Pbqc orf were amplified from P. berghei genomic DNA using primers 9483/9515 and 9516/9486. The primers 9515 and 9516 cover an overlap of a 29 bp region, with the mutations F103A and Q105A that were selected based on studies of active site mutation of QC from X. campestris. These mutations were introduced by site-directed mutagenesis, following the overlap extension PCR. Subsequently, the full-length qcCD, obtained by overlap extension of the two partial fragments using primers 9483/9486, was cloned in pJET 1.2 blunt cloning vector (K1232, Thermo Scientific). This resulted in construct pL2348 that was sequenced to confirm the presence of the two point mutations and the absence of undesired mutations. Transfection of PbΔqc-GIMO parasites with the XhoI and SacII linearized construct pL2348, followed by applying negative selection with 5-fluorocytosine (5-FC), resulted in selection of PbqcCD parasites (line 3276) where the hdhfr::yfcu SM in the qc locus of the PbΔqc-GIMO line is replaced by the mutated Pbqc orf. Correct integration of the constructs into the genome of PbqcCD parasites was analyzed by diagnostic PCR 396 analysis on gDNA and Southern analysis of PFG-separated chromosomes. In addition, a fragment of 624bp was amplified using primers 8991/8992 covering the F103A/Q105A mutated region from PbqcCD gDNA, cloned in pJET 1.2 blunt-end cloning vector and sequenced using pJET forward and reverse primers to confirm the mutations. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | mCherry | ||||||||||||||||||
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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 | This reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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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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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | Luciferase | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) PCR construct 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 | This reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable | ||||||||||||||||||
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 | P230p | ||||||||||||||||||
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