SummaryRMgm-5084
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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) : 34390881 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. yoelii 17XL |
Name parent line/clone | RMgm-5083 |
Other information parent line | The mutant contains a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a promoter. This expression cassette has been introduced in the neutral p230p locus of wild type P. yoelii. This mutant does not contain a drug-selectable marker (SM). The hdhfr-yfcu SM, used to introduce the diCre cassette, has been removed by negative selection. |
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The mutant parasite was generated by | |
Name PI/Researcher | Ishizaki T, Kaneko O |
Name Group/Department | Graduate school of Biomedical Sciences |
Name Institute | Nagasaki University |
City | Nagasaki |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-5084 |
Principal name | PKAc-iKO |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Growth of PKAc-iKO parasites (blood stages) treated with rapamycin (RAP) was significantly lower than those treated with DMSO. The parasitemias of wild type 17XL and cloned DiCre parasites with RAP treatment were not significantly different among all groups, indicating that RAP treatment had no effect on parasite growth. Evidence is presented that (from the Abstract): 'The DiCre-loxP inducible knockout (iKO) system showed more than 80% excision efficacy of the target PKAc locus and more than 90% reduction of locus transcripts 24 hours (one cell cycle) after RAP administration in vivo. Using this developed system, cAMP-dependent protein kinase (PKAc) was inducibly disrupted and we found that PKAc-iKO parasites showed severe growth and erythrocyte invasion defects'. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation In addition, the mutant contains a diCre expression cassette containing the two genes Cre59-FKBP and Cre60-FRB placed on either side of the bidirectional eef1a promoter (see the parent line RMgm-5083 for details about this transgene expression cassette). Protein (function) The rapamycin-inducible Cre recombinase (DiCre) established for use in P. falciparum uses the rapamycin-binding FKBP12 and FRB proteins to dimerise the two enzyme halves. In the DiCre system, Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin‐binding protein (either FKBP12 or FRB, the rapamycin‐binding domain of the FKBP12‐rapamycin‐associated protein mTOR). Rapamycin‐induced heterodimerization of the two components restores recombinase activity (rapamycin‐mediated dimerization of two distinct, enzymatically inactive polypeptides approximately corresponding to the individual domains of Cre (residues Thr19–Asn59, called Cre59, and Asn60‐343, called Cre60) each fused to a different rapamycin‐binding protein (FKBP12 and FRB respectively) results in the reconstitution of Cre recombinase activity). This site–specific recombinase recognizes short, 34 bp sequences called loxP sites and catalyzes the excision or inversion of the floxed (flanked by loxP) DNA segment. After RAP treatment of mice infected with this mutant (to activate Cre recombinase activity): evidence is presented that (from the Abstract): 'The DiCre-loxP inducible knockout (iKO) system showed more than 80% excision efficacy of the target PKAc locus and more than 90% reduction of locus transcripts 24 hours (one cell cycle) after RAP administration in vivo. Using this developed system, cAMP-dependent protein kinase (PKAc) was inducibly disrupted and we found that PKAc-iKO parasites showed severe growth and erythrocyte invasion defects'. . Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_0839000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0934800 | ||||||||||||||||||||||||||
Gene product | cAMP-dependent protein kinase catalytic subunit | ||||||||||||||||||||||||||
Gene product: Alternative name | PKAc; PKA | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | a loxP sequence inserted between PKAc exon 1 and 2 | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||||
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 | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The PKAc 5’- and 3’-HRs were PCR-amplified from parasite gDNA. A DNA fragment containing a loxP intron sequence and recodonized PKAc cDNA sequence corresponding to exons 2 to 5 designed based on the codon usage of P. falciparum (3D7 strain) with the IDT codon optimization tool (https://sg.idtdna.com/CodonOpt; GenScript Biotech, Piscataway, New Jersey, USA) were synthesized. Recodonization was done to avoid unwanted integration to this region in the genome by homologous recombination, instead of the 5’-HR. A DNA fragment containing hDHFR-yFCU open reading frame was PCR-amplified from pDC2-Cas9-PyU6-PypPK1-myc plasmid with primers PKAc-iKOhDHFR/yFCU.F and PKAc-iKO-hDHFR/yFCU.R. All PCR products were purified using a gel extraction kit and ligated into pDC2-Cas9-PyU6-hDHFR plasmid using an In-Fusion HD cloning kit, yielding pDC2-Cas9-PyU6-PKAc-iKO-hDHFR/yFCU. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | diCre (Cre59-FKBP and Cre60-FRB) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||
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 | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | The plasmid pBS_DC_hsp86/Bip5’ was a kind gift from M. Blackman. The promoter to drive FRB-Cre60 and FKBP-Cre59 was replaced with the P. yoelii elongation factor 1 alpha (ef-1α) bi-directional promoter (pBS-DC-Pyef-1α). The DiCre expression cassette was PCR-amplified from pBS-DC-Pyef-1α using oligonucleotide primers DiCre.F and DiCre.R, and ligated into the pDC2-cam-Cas9-PyU6-hDHFR plasmid using an In-Fusion HD cloning kit (Takara Bio Inc., Shiga, Japan), yielding pDC2-Cas9-DC-Pyef-1α. A guide RNA (gRNA) sequence to introduce a double strand break in the p230p locus (PY17X_0306600) was designed using EupaGDT and ligated using T4 ligase (New England Biolabs, Ipswich, MA, USA). The 5’- and 3’- homologous regions (HRs) were PCR-amplified from parasite gDNA with oligonucleotide primers Pyp230p 5HR.F, Pyp230p-5HR.R, Pyp230p-3HR.F, and Pyp230p-3HR.R; and inserted into pDC2-Cas9-DC-Pyef-1α, yielding pDC2-Cas9-p230p-DC-Pyef-1α To generate DiCre-expressing P. yoelii, a DNA fragment containing a DiCre expression cassette and a hDHFR-yFCU expression cassette was integrated into the Pyp230p gene locus, which is dispensable in all development stages. After validating the insertion of this DNA fragment into the target genome locus by genotype PCR, parasites were treated with 5-FC to remove the hDHFR-yFCU expression cassette, then cloned by limiting dilution | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PY17X_1134800 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357000 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | Not available | ||||||||||||||||||
Gene product | Not available | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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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; 230p | ||||||||||||||||||
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