Summary

RMgm-4679
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1400800; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
Transgene
Transgene not Plasmodium: Melanoma-associated antigen 3 (MAGE-A3)
Promoter: Gene model: PBANKA_1400800; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
3'UTR: Gene model: PBANKA_1400800; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
Replacement locus: Gene model: PBANKA_1400800; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
Phenotype Sporozoite; Liver stage;
Last modified: 23 September 2019, 11:22
  *RMgm-4679
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherZhou D, Dai J
Name Group/DepartmentDepartment of Thoracic Surgery, Xinqiao Hospital
Name InstituteThird Military Medical University
CityChongqing
CountryChina
Name of the mutant parasite
RMgm numberRMgm-4679
Principal nameGAS/MAGE-A3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNormal numbers of salivary gland sporozoites produced.
Liver stageImmunofluorescence 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
The mutant lacks expression of UIS3 and expresses Melanoma-associated antigen 3 (MAGE-A3) under control of the uis3 promoter

The gene has been disrupted using CRISPR/cas9 genome editing (using constructs as described for mutant
RMgm-1095).

Protein (function)
UIS3
A protein belonging to the ETRAMP family of proteins. It is expressed (transcribed) in sporozoites and expressed in liver stages and localizes to the parasitophorous vacuole. Disruption of the uis3 gene results in early arrest of liver stage development.

Melanoma-associated antigen 3 (MAGE-A3):
This protein is a member of the cancer testis antigen (CTA) family, is highly expressed in non-small cell lung cancers (NSCLCs). MAGE-A3-based anti-lung cancer immunotherapies are currently being developed. 

Phenotype
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. These findings indicate that GAS could successfully deliver the lung-cancer antigen, MAGE-A3, in HepG2 cells.

Additional information
Disruption of the uis3 gene results in early arrest of liver stage development. In this study mice were immunized with attenuated GAS/MAGE-A3 sporozoites (immunized intravenously three times at 2-week intervals with either phosphate-buffered saline (PBS) or 5 × 104 GAS/MAGE-A3 sporozoites). Evidence is presented for the following:
- GAS/MAGE-A3 triggers a robust and specific CD8+ T cell response in HLA-A2 transgenic mice 
- Adoptive immunotherapy using CD8+ T cells suppresses subcutaneous A549 cell tumor growth in nude mice

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1400800
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThis 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  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameMelanoma-associated antigen 3 (MAGE-A3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1400800
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_1400800
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_1400800
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4