RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5150
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1 (AMA1)
Details mutation: The mutant contains a mutated ama1 gene with two LoxN sites (up- and downstream of the gene)
Transgene
Transgene not Plasmodium: DiCre
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): heat shock protein 70; Gene product: HSP70
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 23 June 2022, 16:35
  *RMgm-5150
Successful modificationThe 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) : 35731833
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, the mutant has the reporter gene mCherry in the 230p locus (under the hsp70 promoter). The mutant does not contain a drug-selectable marker
The mutant parasite was generated by
Name PI/ResearcherFernandes P, Silvie O
Name Group/DepartmentCentre d'Immunologie et des Maladies Infectieuses
Name InstituteINSERM, CNRS, CIMI-Paris, Sorbonne Université
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-5150
Principal nameama1cKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
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

Mutant/mutation
The mutant contains a mutated ama1 gene with two LoxN sites, one upstream and one downstream of the gene. It also contains a GFP expression cassette, upstream of ama1 but between the two LoxN sites.
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/10.1101/2022.01.04.474787  

Protein (function)

The micronemal protein AMA1 is a type I transmembrane protein and is a surface protein of the merozoite. Merozoites invade target host cells actively by gliding through a structure known as the moving junction (MJ). formation of the MJ involves the export of rhoptry neck proteins RONs into the host cell, where RON2 is inserted into the host cell membrane and serves as a receptor for the Apical Membrane Antigen 1 (AMA1), that is secreted from the micronemes onto the surface of the parasite. Formation of the MJ is associated with active penetration inside the parasitophorous vacuole (PV), which is essential for further development and replication of the parasite.

Phenotype
No phenotype different from wildtype parasites.
After rapamycin treatment of parasites using methods described for mutant RMgm-4900, the ama1 gene (and the GFP gene) is excised. See Additional Information below. 

Additional information

From the abstract:
' In this study, we used the DiCre system to achieve conditional deletion of ama1, ron2 and ron4 genes in P. berghei sporozoites. Our data reveal that sporozoites rely on AMA1 and RONs to invade salivary glands in the mosquito and hepatocytes in the mammalian host, implying a conserved feature of the invasion process across invasive stages of Plasmodium.'

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative nameAMA1
Details of the genetic modification
Short description of the mutationThe mutant contains a mutated ama1 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 modificationIn order to target different genes of interest, we first generated a generic plasmid, pDownstream1Lox (Addgene #164574), containing a GFP-2A-hDHFR cassette under the control of a P. yoelii hsp70 promoter and followed by the 3’UTR of P. berghei calmodulin (cam) gene and a single LoxN site. The plasmid also contains a yFCU cassette to enable the elimination of parasites carrying episomes by negative selection with 5-fluorocytosine. The ama1Con plasmid was designed to excise only ~30 bp downstream of P. berghei ama1 3’UTR. Two fragments were inserted on each side of the GFP-2A-hDHFR cassette of the pDownstream1Lox plasmid: a 5’ homology region (HR) homologous to the terminal portion of ama1 (ORF and 3’ UTR) followed by a single LoxN site, and a 3’ HR homologous to a sequence downstream of the 3’ UTR of ama1 gene. The ama1Dutr plasmid was assembled similarly to the ama1Con construct except that the 5’ HR consisted in the terminal portion of ama1 ORF followed by a LoxN site and the 3’ UTR of P. yoelii ama1, to allow excision of the 3’UTR upon rapamycin activation of DiCre. The ama1cKO plasmid was designed to introduce a single LoxN site upstream of ama1 in the ama1Conrapa parasites, which already contained a residual LoxN site downstream of the gene. To generate the ama1cKO plasmid, the pDownstream1Lox vector was first modified to remove the downstream LoxN site. Then, a 5’ HR and a 3’ HR, both homologous to sequences located upstream of ama1 gene, were cloned into the modified plasmid on each side of the GFP-2A-hDHFR, with a single LoxN site introduced upstream of the GFP-2A-hDHFR cassette. To generate ron2cKO and ron4cKO constructs, two separate plasmids, P1 and P2, were generated to insert a LoxN site upstream of the promoter and downstream of the gene of interest, respectively, in two consecutive transfections. P1 plasmids were constructed by insertion of 5’ and 3’ HR on each side of the GFP-2A-hDHFR cassette in the pDownstream1Lox plasmid, with a second LoxN site introduced upstream of the GFP cassette. The 5’ HR and 3’ HR correspond to consecutive fragments located in the promoter region of the GOI. Because the intergenic sequence between ron4 gene and its upstream gene is short, and in order to maintain expression of the upstream gene and exclude any unwanted duplication and spontaneous recombination events, we introduced the 5’ HR of ron4 in two fragments, with fragment 1 corresponding to the region just upstream of the ORF while fragment 2 corresponded to the 3’ UTR from the P. yoelii ortholog of the upstream gene. P2 plasmids were constructed in a similar manner by insertion of a 5’ HR and a 3’HR on each side of the GFP-2A-hDHFR cassette in the pDownstream1Lox plasmid. The 3’ HR regions corresponded to the 3’ UTR sequences of RON2 or RON4, respectively. For both target genes, the 5’ HR was divided into two fragments, where fragment 1 corresponded to the end of the ORF followed by a triple Flag tag, and fragment 2 corresponded to the 3’ UTR from the P. yoelii ortholog gene, in order to avoid duplication of the 3’ UTR region and spontaneous recombination
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
Details of the genetic modification
Inducable system usedNo
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 parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
Details of the genetic modification
Inducable system usedNo
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 parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog heat shock protein 70
Gene productHSP70
Gene product: Alternative name
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 PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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