RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_0514200; Gene model (P.falciparum): PF3D7_1030200; Gene product: claudin-like apicomplexan microneme protein, putative (CLAMP)
Details mutation: a mutated clamp gene with two LoxN sites (up- and downstream of the gene)
PhenotypeNo phenotype has been described
Last modified: 21 March 2023, 10:48
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36928686
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4900
Other information parent lineThe mutant RMgm-4900 contains a DiCre expression cassette. The Dicre gene (the N-terminal Cre 59 (residues Thr19-Asn59) and C-terminal Cre 60 (Asn60-Asp343) portions of the Cre fused at their N-terminus to FKBP12 and FRB, respectively) is under control of the constitutive hsp70 promoter. The DiCre expression cassette is introduced into the silent p230p locus. In addition, it expresses mCherry and does not contain a drug-selectable marker
The mutant parasite was generated by
Name PI/ResearcherLoubens M, Silvie O
Name Group/DepartmentSorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses
Name InstituteCIMI-Paris
Name of the mutant parasite
RMgm numberRMgm-5253
Principal nameclampcKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

The mutant contains a mutated clamp gene with two LoxN sites, one upstream and one downstream of the gene. It also contains a gfp and hdhfr expression cassette, upstream of the clamp gene but between the two LoxN sites. The clamp open reading frame (ORF) is C-terminally tagged with a triple Flag epitope.
In addition, it contains a Dicre expression cassette and an mCherry expression cassette, both introduced into the neutral 230p locus.
Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.08.29.505663

Protein (function)
Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion in Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. Topology predictions indicate structural similarities between CLAMP and the mammalian tight-junction proteins claudin-15 and claudin-19. CLAMP is also expressed in Plasmodium sporozoites, the mosquito transmitted forms of the malaria parasite. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion.

No phenotype different from wildtype parasites.
After rapamycin treatment of parasites the clamp gene (and the GFP gene) is excised. See Additional Information below. 

Additional information
'We have previously implemented the DiCre system in the rodent malaria parasite P. berghei, and showed that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle'.
In this study, we used the DiCre system in P. berghei to study the role of CLAMP in sporozoites. Conditional deletion of clamp gene resulted in a dramatic decrease of sporozoite invasion of the mosquito salivary glands and of mammalian hepatocytes. This severe phenotype was associated with a major defect in sporozoite gliding motility and reduced shedding of TRAP.'

Evidence is presented that:
- Rapamycin-induced gene excision abrogates CLAMP protein expression in sporozoites
- CLAMP is essential for sporozoite cell traversal and hepatocyte invasion 
- CLAMP is essential for sporozoite gliding motility
- CLAMP is required for TRAP shedding
- CLAMP is localized in a subset of micronemes and accumulates at the apical tip of sporozoites

Other mutants

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0514200
Gene Model P. falciparum ortholog PF3D7_1030200
Gene productclaudin-like apicomplexan microneme protein, putative
Gene product: Alternative nameCLAMP
Details of the genetic modification
Short description of the mutationa mutated clamp gene with two LoxN sites (up- and downstream of the gene)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
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
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTwo plasmids, P1 and P2, were generated for insertion of LoxN sites upstream and downstream of clamp, respectively. The P1 plasmid was assembled by inserting two homologous sequences, 5’HR1 and 5’HR2, both localized upstream 463 of clamp promoter region, in the pUpstream2Lox plasmid (Addgene #164573), which contains a GFP-2A-hDHFR cassette flanked by two LoxN sites. Both 5’HR1 (895 bp) and 5’HR2 (1014 bp) were amplified by PCR from WT PbANKA genomic DNA, and inserted into KpnI/XhoI and NheI sites, respectively, of the pUpstream2Lox plasmid. The P2 plasmid was assembled by inserting two homologous sequences, 3’HR1 and 3’HR2, corresponding to the end of clamp ORF and to clamp 3’UTR, respectively, in the pDownstream1Lox plasmid (Addgene #164574), which contains a GFP-2A-hDHFR cassette followed by a single LoxN site. Both 3’HR1 (862 bp) and 3’HR2 (999 bp) were amplified by PCR from WT PbANKA genomic DNA, and inserted into KpnI/XhoI and NheI sites, respectively, of the pDownstream1Lox plasmid. A triple Flag epitope tag was inserted in frame with clamp ORF immediately before the stop codon. In addition, a 559 bp fragment corresponding to the 3’ UTR sequence from P. yoelii clamp gene (PY17X_0515300) was inserted immediately downstream of 3’HR1, to allow proper gene expression and avoid spontaneous recombination with the 3’ UTR of P. berghei clamp, which was used as 3’HR2. Plasmids P1 and P2 were verified by Sanger DNA sequencing (Eurofins Genomics) and linearized with KpnI and NheI before transfection. Parental DiCre parasites were transfected with the P1 plasmid to generate clamp-P1 parasites, which were then exposed to rapamycin and transfected with the P2 plasmid to generate the final clampcKO line For the first transfection, schizonts purified from an overnight culture of PbDiCre blood stage parasites were transfected with 10 μg of linearized P1 plasmid using the AMAXA Nucleofector device (program U033), and immediately injected intravenously into the tail vein of SWISS mice. To permit the selection of resistant transgenic parasites, pyrimethamine (35 mg/L) and 5-flurocytosine (0.5 mg/ml) were added to the mouse drinking water, starting one day after transfection. The parasitaemia was monitored daily by flow cytometry and the mice sacrificed at a parasitaemia of 2-3%, allowing preparation of frozen parasite stocks and isolation of parasites for genomic DNA extraction. After drug selection, GFP+/mCherry+ clamp-P1 parasites were sorted by flow cytometry. Mice were injected intraperitoneally 491 with frozen
492 parasite stocks and monitored until the parasitaemia was between 0.1 and 1%. On the day of sorting, one drop of tail blood was collected in 1ml PBS and used for sorting of 100 iRBCs on a FACSAria II (Becton-Dickinson). Sorted parasites were recovered in 200 μl RPMI containing 20% FBS and injected intravenously into two mice. clamp-P1 parasites were exposed to a single dose of rapamycin to induce excision of the GFP-2A-hDHFR cassette. The resulting GFP-/mCherry+ clamp-P1 parasites, which retained a single LoxN site inserted upstream of clamp, were sorted by flow cytometry and amplified in mice.
Rapamycin-exposed clamp-P1 parasites were then transfected with the P2 plasmid to generate the clampcKO line. GFP+/mCherry+ clampcKO parasites were sorted by flow cytometry and cloned by limiting dilution and injection into mice to generate the final population.
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