SummaryRMgm-5150
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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, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 35731833 |
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 | RMgm-4900 |
Other information parent line | The 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 |
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The mutant parasite was generated by | |
Name PI/Researcher | Fernandes P, Silvie O |
Name Group/Department | Centre d'Immunologie et des Maladies Infectieuses |
Name Institute | INSERM, CNRS, CIMI-Paris, Sorbonne Université |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-5150 |
Principal name | ama1cKO |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation From the abstract: 'Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Three-dimensional electron microscopy data showed that sporozoites enter salivary gland cells through a ring-like structure and by forming a transient vacuole. The absence of a functional AMA1-RON complex led to an altered morphology of the entry junction, associated with epithelial cell damage. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to efficiently and safely enter the mosquito salivary glands to ensure successful parasite transmission'. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0915000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1133400 | ||||||||||||||||||||||||||
Gene product | apical membrane antigen 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | AMA1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The mutant contains a mutated ama1 gene with two LoxN sites (up- and downstream of the gene) | ||||||||||||||||||||||||||
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 | 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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | In 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 | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | DiCre | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
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 | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | mCherry | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | heat shock protein 70 | ||||||||||||||||||
Gene product | HSP70 | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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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 | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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