RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5084
Malaria parasiteP. yoelii
Genotype
MutatedGene model (rodent): PY17X_0839000; Gene model (P.falciparum): PF3D7_0934800; Gene product: cAMP-dependent protein kinase catalytic subunit (PKAc; PKA)
Details mutation: a loxP sequence inserted between PKAc exon 1 and 2
Transgene
Transgene not Plasmodium: diCre (Cre59-FKBP and Cre60-FRB)
Promoter: Gene model: PY17X_1134800; Gene model (P.falciparum): PF3D7_1357000; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230p)
Phenotype Asexual bloodstage;
Last modified: 17 August 2021, 13:18
  *RMgm-5084
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. yoelii 17XL
Name parent line/clone RMgm-5083
Other information parent lineThe 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.
The mutant parasite was generated by
Name PI/ResearcherIshizaki T, Kaneko O
Name Group/DepartmentGraduate school of Biomedical Sciences
Name InstituteNagasaki University
CityNagasaki
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5084
Principal namePKAc-iKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGrowth 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/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the PKAc gene (cAMP-dependent protein kinase) of P. yoelii with a mutated PKAc gene that has a loxP sequence inserted between PKAc exon 1 and 2.

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)
cAMP-dependent protein kinase (evidence has been presented that it phosphorylates Ser610 of P. falciparum AMA1, which is essential for erythrocyte invasion). 

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.

Phenotype
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.

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'. .

Additional information
About rapamycin (RAP) treatment: ' RAP (Sigma-Aldrich) was dissolved in DMSO (4 mg/mL) and stored at ‒20°C until use. Schizont stage parasites were enriched using Nycodenz (1.077 g/mL; company info) and incubated at 15°C for 3 hours until their maturation. Matured schizonts were intravenously inoculated into mice and 4 mg/kg RAP resuspended in PBS was intraperitoneally administrated. Blood was collected from 3 mice at 3, 6, 12, and 24 hours after RAP administration.'

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0839000
Gene Model P. falciparum ortholog PF3D7_0934800
Gene productcAMP-dependent protein kinase catalytic subunit
Gene product: Alternative namePKAc; PKA
Details of the genetic modification
Short description of the mutationa loxP sequence inserted between PKAc exon 1 and 2
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namediCre (Cre59-FKBP and Cre60-FRB)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe 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
Other details transgene
Promoter
Gene Model of Parasite PY17X_1134800
Gene Model P. falciparum ortholog PF3D7_1357000
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p; 230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4