SummaryRMgm-952
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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) : 24089525 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei NK65 |
Name parent line/clone | RMgm-269 |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | L. Tawk, R. Menard, JC Barale |
Name Group/Department | Unité de Biologie et Génétique du Paludisme |
Name Institute | Institut Pasteur |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-952 |
Principal name | SUB1/Cond |
Alternative name | Clone SUB1/Cond-5 |
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 | PCR analysis of genomic DNA of the mutant sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1. The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced liver stages at 24 and 48 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number and size to that of wild type sporozoites. Evidence is presented for normal development of liver merozoites but a reduced egress of liver merozoites from the mature liver schizonts. Evidence is presented that the reduced egress is due to lack of rupture of the PVM indicating a role of SUB1 in PVM rupture. in addition, evidence is presented for a role of SUB1 in processing of SERA3 and MSP1. SUB1-deficient liver stage merozoites are unable to establish a blood stage infection. |
Additional remarks phenotype | Mutant/mutation PCR analysis of genomic DNA of the mutant sporozoites showed high levels (>90%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed ORF of SUB1. Additional information Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1107100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0507500 | ||||||||||||||||||||||||||
Gene product | subtilisin-like protease 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | SUB1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The mutant contains a FRTed ORF of the sub1 locus | ||||||||||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | The ORF of sub1 is removed in the sporozoite stage by the Flp/FRT system. | ||||||||||||||||||||||||||
Additional remarks inducable system |
![]() ![]() This mutant has been generated by replacement of the endogenous sub1 gene by an sub1 gene with a FRTed ORF in mutant RMgm-269 that expresses Flp and GFP. Removal of the FRTed ORF of sub1 has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the ORF of sub1 which was flanked by FRT sequences. | ||||||||||||||||||||||||||
Type of plasmid/construct | 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 | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | WR99210 | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The plasmid pSUB1/FRT was designed to place, following double cross-over recombination, a 5’ FRT site 736 bp upstream from the sub1 start codon and a 3’ FRT site downstream from the 3’ UTR of sub1 and the selectable marker. The targeting pSUB1/FRT plasmid was constructed by cloning three PCR products into a plasmid that carries two Flp recognition target (FRT) sites and the human dihydrofolate reductase (hDHFR) expression cassette i) a 736 bp fragment upstream the Pbsub1 locus; ii) a 3479bp DNA fragment encompassing the 5’ FRT site, 1177bp corresponding to the 5’ UnTranslated Region (UTR) of Pbsub1, the complete PbSUB1 open reading frame (1799 bp) and 503 bp corresponding to Pbsub1 3’ UTR and iii) a 673 bp fragment downstream the Pbsub1 locus. The latter was amplified using the oligonucleotides 5’-CCCGGGCATTTGGTGTATATTATTGTTTTTCTTG-3’ and 5’-GGTACCTCAATGTGCATTTATTGTTATGC-3’ and was cloned first in the plasmid using SmaI and KpnI restriction sites. To the resulting plasmid was added the 736 bp fragment upstream the Pbsub1 locus amplified using the primers 5’-AAGCTTTAAATAAATCACCAAATATTA AATTGG-3’ and 5’-GCGGCCGCATGTGTGTTATATAAGTGATTATTCTTTAAGC-3’ using HindIII and NotI restriction sites. Finally, the entire Pbsub1 locus was amplified using the 5’-GATATCTAAATCGAAGAAAATATAAACATTCG-3’ and 5’-GGGCCCAAATATATGTCGGTGTAATGCACC-3’ primers and cloned using the EcoRV and ApaI restriction sites. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | Flp recombinase of yeast (Flp) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||
Additional remarks inducable system | The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. | ||||||||||||||||||
Type of plasmid/construct | Plasmid single 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 | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The flpl gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. In the resulting mutant (UIS4/Flp(-), by integrating at the DHFR-TS locus, a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains the pyrimethamine resistant pbdhfr-ts gene. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
Gene Model P. falciparum ortholog | Not available | ||||||||||||||||||
Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | 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 | pbdhfr | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The flpl gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. In the resulting mutant (UIS4/Flp(-), by integrating at the DHFR-TS locus a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene. To make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant pbdhfr-ts gene | ||||||||||||||||||
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 | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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