SummaryRMgm-4552
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption, Gene mutation, Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30315162 |
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 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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The mutant parasite was generated by | |
Name PI/Researcher | Fang H, Billker O, Brochet M |
Name Group/Department | Department of Microbiology and Molecular Medicine, Faculty of Medicine |
Name Institute | University of Geneva |
City | Geneva |
Country | Switzerland |
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Name of the mutant parasite | |
RMgm number | RMgm-4552 |
Principal name | CDPK4-KO/pkg(T619Q)-3xHA |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Severely reduced growth rate of asexual blood stages. From the Abstract: Most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Cyclic GMP-dependent protein kinases (PKGs) are the major mediators of the cGMP signal transduction pathway and regulate a variety of physiological effects. PKG is is a serinethreonine kinase that transfers the γ-phosphate of ATP, in a cGMP-dependent manner, to a variety of substrate proteins. Evidence has been presented that in Plasmodium activation of PKG is critically required to regulate cytosolic Ca2+ levels in multiple life cycle stages. PKG emerges as a universal regulator that controls ookinete gliding, gametocyte activation, and schizont rupture. Phenotype To verify the negative interaction between pkg and cdpk4, a cdpk4-KO/pkgT619Q-3xHA clonal line was generated and found to have severely reduced growth if compared to either of the single mutants. Additional information |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0615200 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0717500 | ||||||||||||||||||||||||
Gene product | calcium-dependent protein kinase 4 | ||||||||||||||||||||||||
Gene product: Alternative name | CDPK4 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) PCR construct double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | Yes | ||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | 087803 | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||
Additional remarks genetic modification | Preparation of targeting vectors. 3xHA tagging, knockout and allelic replacement constructs in P. berghei were generated using phage recombineering in Escherichia coli tryptic soy agar (TSA) bacterial strain with PlasmoGEM vectors (http://plasmogem.sanger.ac.uk/). For final targeting vectors not available in the PlasmoGEM repository, generation tagging constructs was performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R for 3xHA tagging. Substitution of the GAP40(S148/149A) residue was introduced using primer gap40S148 HA-F instead of gap40 HA-F. Mutations were confirmed by sequencing with primers gap40-QCR1 and GW1. The modified library inserts were released from the plasmid backbone using NotI. | ||||||||||||||||||||||||
Additional remarks selection procedure | The mutant does not contain the hdhfr/yfcu drug-selectable marker cassette. This cassette has been removed by negative selection. | ||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1008200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1436600 | ||||||||||||||||||||||||||
Gene product | cGMP-dependent protein kinase | ||||||||||||||||||||||||||
Gene product: Alternative name | PKG | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | a mutated pkg in which the threonine gatekeeper residue ((T619Q) is mutated to glutamine | ||||||||||||||||||||||||||
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 | Yes | ||||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | - | ||||||||||||||||||||||||||
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 | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | Point mutations were introduced with a two-step strategy using l Red-ET recombineering in E. coli. The first step involved the insertion by homologous recombination of a Zeocin-resistance/Phe-sensitivity cassette surrounded by sequences 5' and 3' of the codon of interest amplified using primer pairs specific for the gene of interest (GOI): goi-delF/goi-delR. Recombinant bacteria were then selected on Zeocin. After verification of the recombination event by PCR, a second round of recombination exchanged the Zeocin-resistance/Phe-sensitivity cassette with a PCR product containing the desired mutation surrounding the codon of interest amplified using goi-mutF/goi-mutR primer pairs. Bacteria were selected on YEG-Cl kanamycin plates. Mutations were confirmed by sequencing vectors isolated from colonies sensitive to Zeocin. Generation of knockout and tagging constructs was performed using sequential recombineering and gateway steps as previously described. Lambda red-ET recombineering was first used to introduce a bacterial selection marker amplified into the gDNA insert, such that the target gene is either deleted or prepared for 39-end tagging. The bacterial marker was then replaced with a selection cassette for P. berghei in a Gateway LR Clonase reaction in vitro. The modified library inserts were then released from the plasmid backbone using NotI and used to transfect P. berghei. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1008200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1436600 | ||||||||||||||||||||||||||
Gene product | cGMP-dependent protein kinase | ||||||||||||||||||||||||||
Gene product: Alternative name | PKG | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | triple-HA | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | Yes | ||||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | - | ||||||||||||||||||||||||||
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 | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | No details are provided for the PlasmoGEM vector used | ||||||||||||||||||||||||||
Additional remarks selection procedure | The mutant does not contain a selectable marker. The positive/negative selectable marker cassette has been removed by negative selection with 5-FC. | ||||||||||||||||||||||||||
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