SummaryRMgm-4679
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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 |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 31516749 |
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 | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Zhou D, Dai J |
Name Group/Department | Department of Thoracic Surgery, Xinqiao Hospital |
Name Institute | Third Military Medical University |
City | Chongqing |
Country | China |
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Name of the mutant parasite | |
RMgm number | RMgm-4679 |
Principal name | GAS/MAGE-A3 |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Normal numbers of salivary gland sporozoites produced. |
Liver stage | Immunofluorescence and Western blot analyses performed 24 h after GAS/MAGEA3 sporozoite invasion into HepG2 cells showed that MAGEA3 was expressed in GAS/MAGE-A3-infected cells. |
Additional remarks phenotype | Mutant/mutation Protein (function) Melanoma-associated antigen 3 (MAGE-A3): Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1400800 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1302200 | ||||||||||||||||||||||||
Gene product | early transcribed membrane protein 13 | ||||||||||||||||||||||||
Gene product: Alternative name | UIS3; up-regulated in infective sporozoites; ETRAMP13 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
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 | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | This uis3 gene has been disrupted by the CRISPR/Cas9 genome editing system through introduction of a double strand break (by Cas9 and a targeting single guide RNA; sgRNA), followed by repair through homologous recombination (see RMgmDB-1095). In addition, the MAGE-A3 gene is at the same time introduced into the disrupted uis3 locus. Recombinant parasites were constructed by using CRISPRCas9 as previously described (RMgm-1095). Plasmid pYC expressing DNA endonuclease enzyme (Cas9) and pyrimethamine resistance genes (a gift from Professor Yuan Jing of Xiamen University) was used to construct a genetically attenuated, MAGE-A3-expressing sporozoite (GAS/MAGE-A3). First, sgRNA (5’-GCAATTGCTTTGTTATCATC-3’), specifically targeting P.b. ANKA UIS3 gene, was inserted downstream to the Plasmodium U6 promoter in the pYC plasmid. Second, the homologous recombinant fragment for replacing the whole UIS3 coding sequence (856 bp) containing a 5’UTR of UIS3 [coding sequence of human MAGE-A3 (900 bp)] and a 3’UTR of UIS3 was constructed by overlapping PCR and inserted into multiple clone sites of the pYC plasmid | ||||||||||||||||||||||||
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 | Melanoma-associated antigen 3 (MAGE-A3) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||
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 | |||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1400800 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1302200 | ||||||||||||||||||
Gene product | early transcribed membrane protein 13 | ||||||||||||||||||
Gene product: Alternative name | UIS3; up-regulated in infective sporozoites; ETRAMP13 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1400800 | ||||||||||||||||||
Gene product | early transcribed membrane protein 13 | ||||||||||||||||||
Gene product: Alternative name | UIS3; up-regulated in infective sporozoites; ETRAMP13 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_1400800 | ||||||||||||||||||
Gene product | early transcribed membrane protein 13 | ||||||||||||||||||
Gene product: Alternative name | UIS3; up-regulated in infective sporozoites; ETRAMP13 | ||||||||||||||||||
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