SummaryRMgm-5070
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption, Introduction of a transgene, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 36634679 |
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 | P. berghei ANKA 820cl1m1cl1 (RMgm-164) |
Other information parent line | P. berghei ANKA 820cl1m1cl1 (RMgm-164) is a reference ANKA mutant line which expresses GFP under control of a male and RFP under control of a female gametocyte specific promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 19438517). |
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The mutant parasite was generated by | |
Name PI/Researcher | Russell AJC, Billker O |
Name Group/Department | Department of Molecular Biology |
Name Institute | Umeå University |
City | Umeå |
Country | Sweden |
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Name of the mutant parasite | |
RMgm number | RMgm-5070 |
Principal name | fd2-KO (cl1, cl3) |
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 | Infertile females, fertile males. |
Fertilization and ookinete | Infertile females, fertile males. Complete (or nearly complete) loss in the ability to form oocysts in mosquitoes |
Oocyst | Complete (or nearly complete) loss in the ability to form oocysts in mosquitoes |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotypes of 10 gene-deletion mutants: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0902300 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1146800 | ||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | Yes | ||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | PbGEM-287226 | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | Most gene targeting vectors were obtained from the PlasmoGEM resource (http://plasmogem.sanger.ac.uk/search) and most vectors carried the default PlasmoGEM 3xHA-hdhfr-yfcu gene replacement cassette. Since PlasmoGEM vectors were not available to target md1 and fd1, knock-out vectors were prepared using PCR. For md1 amplicons of 1.25 kb upstream and downstream of the gene were amplified from genomic DNA and then assembled either side of a selection cassette amplified from the PlasmoGEM 3xHA-hdhfr-yfcu gateway vector by Gibson Assembly using primer overhangs. The Gibson product was used as the input for a PCR reaction to amplify the entire construct, which was then gel-purified prior to transfection. For fd1 a CRISPR/Cas9 knock-out vector was constructed by PCR amplification of 0.5 kb 5’ and 3’ regions of the coding sequence of the target gene from genomic DNA, which were restrictionligation cloned so to flank an eef1ɑ 5’UTR-tgdhfr-CAM 3’UTR resistance cassette in a vector also holding the U6 RNA Polymerase 3 promoter from Plasmodium yoelii to drive expression of the target specific guide RNA (gRNA) cloned in by BsmBI, to generate plasmid ABR063. The fd1 CRISPR/Cas9 knock-out line was generated in a split-Cas9 line where conditional activation of CAS9 is achieved by rapamycin-induced dimerisation of a C-terminal fragment of Cas9 fused to FK506- and rapamycin-Binding Protein domain (C-Cas9-FKBP) and a N-terminal fragment of Cas9 fused to the FKBP-Rapamycin Binding domain (N-Cas9-FRB). To achieve this line, N-Cas9-FRB and C-Cas9-FKBP was cloned so to flank the bidirectional 5’UTR of eef1ɑ, and to become nested within 5’ and 3’ targeting sites for the p230p locus generating plasmid ABR010. For scRNA sequencing experiments, where possible, FACS sortable knockout vectors were generated by converting PlasmoGEM intermediate vectors into knockout vector with a gateway cassette containing an pbhsp70 5’utr-GFPmut3-pbdhfr 3’utr-hdhfr-yfcu expression cassette for constitutive expression of GFP upon vector integration, which allows for FACS sorting to select wild-type free ko parasites without the need for dilution cloning. The gateway construct was generated by amplifying a 1.4 kb fragment of the 5’utr of hsp70 from P. berghei genomic DNA , which was cloned into the R6K-GFPmut3-hdhfr-yfcu Gateway vector (added to the PlasmoGEM tagging vector repertoire), upstream of GFPmut3 to generate the R6K-hsp70p-GFPmut3-hdhfr-yfcu Gateway vector. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | RFP | ||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
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_1319500 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1455800 | ||||||||||||||||||
Gene product | LCCL domain-containing protein | ||||||||||||||||||
Gene product: Alternative name | LAP4; LCCL/lectin adhesive-like protein 4; CCp2 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1359600 | ||||||||||||||||||
Gene product | transmission blocking target antigen precursor 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | P48/45 | ||||||||||||||||||
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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 (gfp-mut3) | ||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
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_0416100 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0905300 | ||||||||||||||||||
Gene product | dynein heavy chain, putative | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1010600 | ||||||||||||||||||
Gene product | calmodulin, putative | ||||||||||||||||||
Gene product: Alternative name | cam | ||||||||||||||||||
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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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