SummaryRMgm-4574
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Introduction of a transgene, Introduction of a transgene, Introduction of a transgene |
Reference (PubMed-PMID number) | Not published (yet) |
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-4572 |
Other information parent line | This GIMO line contains a hdfr/yfcu selection marker (SM) cassette in the neutral s1 (PBANKA_1206800) gene locus. In addition, this line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. |
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The mutant parasite was generated by | |
Name PI/Researcher | Halbroth BR, Spencer AJ, Janse CJ, Khan SM, Salman AM |
Name Group/Department | The Jenner Institute |
Name Institute | University of Oxford |
City | Oxford |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-4574 |
Principal name | 2398 cl4 |
Alternative name | PfTRAP+PfLSAP2 |
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 |
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: Plasmodium | ||||||||||||||||||
Gene Model of Parasite | PF3D7_1335900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||
Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||
Gene product: Alternative name | TRAP; SSP2 | ||||||||||||||||||
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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 | No selectable marker | ||||||||||||||||||
Promoter of the selectable marker | No | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | We replaced the positive-negative SM in the genome of the PbANKA-PfTRAP+PbΔs1 line (line 2353 cl3) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSAP2 and PfTRAP. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSA1 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection. The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4572. This parent line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. | ||||||||||||||||||
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 | ETRAMP10.3 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
Gene product: Alternative name | ETRAMP10.3 | ||||||||||||||||||
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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 | GFP-Luciferase | ||||||||||||||||||
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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 | No selectable marker | ||||||||||||||||||
Promoter of the selectable marker | No | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | WWe replaced the positive-negative SM in the genome of the PbANKA-PfTRAP+PbΔs1 line (line 2353 cl3) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSAP2 and PfTRAP. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSA1 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection. The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4572. This parent line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. | ||||||||||||||||||
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 | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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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: Plasmodium | ||||||||||||||||||
Gene Model of Parasite | Not available | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0202100 | ||||||||||||||||||
Gene product | liver stage associated protein 2; Plasmodium exported protein (PHISTc), unknown function | ||||||||||||||||||
Gene product: Alternative name | LSAP2 | ||||||||||||||||||
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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 | No selectable marker | ||||||||||||||||||
Promoter of the selectable marker | No | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | We replaced the positive-negative SM in the genome of the PbANKA-PfTRAP+PbΔs1 line (line 2353 cl3) with the PfLSAP2 CDS by GIMO transfection to create a P. berghei DAG chimeric line expressing both PfLSAP2 and PfTRAP. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with the PfLSAP2 CDS expression cassette, generating plasmid pL2044. The expression cassette in pL2044 contained the PfLSAP2 CDS flanked by the 5′ and 3′ regulatory sequences of Pbuis4, which were amplified from P. berghei ANKA WT genomic DNA. The PfLSAP2 CDS was amplified from genomic DNA of the NF54 strain of P. falciparum. The pL2044 construct was sequenced to ensure that there were no mutations in the PfLSA1 CDS. The construct was linearized using the HindIII restriction enzyme sites outside the 5′ and 3′ Pbs1 TRs before transfection. The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4572. This parent line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. | ||||||||||||||||||
Additional remarks selection procedure | The PfLSAP2 expression cassette has been introduced into the neutral s1 locus by GIMO transfection in GIMO parent line RMgm-4572. This parent line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. | ||||||||||||||||||
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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 | ETRAMP10.3 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0501200 | ||||||||||||||||||
Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||
Gene product: Alternative name | ETRAMP10.3 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_1206800 | ||||||||||||||||||
Gene product | zinc finger (CCCH type) protein, putative | ||||||||||||||||||
Gene product: Alternative name | s1 | ||||||||||||||||||
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