RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1248
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c- or d-type unit))
Phenotype Asexual bloodstage; Sporozoite; Liver stage;
Last modified: 2 May 2015, 14:24
  *RMgm-1248
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25859153
MR4 number
top of page
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
top of page
The mutant parasite was generated by
Name PI/ResearcherMatsuoka H; Hirai M
Name Group/DepartmentDivision of Medical Zoology
Name InstituteJichi Medical University
CityYakushiji, Shimotsuke-shi
CountryJapan
top of page
Name of the mutant parasite
RMgm numberRMgm-1248
Principal namePbLuc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
Phenotype
Asexual blood stageLuciferase expression in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteLuciferase expression in sporeozoites
Liver stageLuciferase expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses Luciferase under the control of the constitutive eef1a promoter. The transgene is integrated into a small subunit ribosomal rna gene (c- or d-type unit; which may affect oocyst/sporozoite production slightly) by single cross-over integration using an insertion construct. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.

Similar mutants expressing luciferase has been reported previously; also mutants with luciferase integrated by double cross-over integration into a silent locus.

  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
top of page
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid single cross-over
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe pGL4.11 luciferase reporter vector (Promega Co., Madison, WI, USA) was purchased. The firefly luciferase gene was amplified by PCR using a pair of primers (Luc-BamF: ggggatccATGGAAGACGCCAAAAACATAAAG and Luc-BamR: ggggatccTTACACGGCGATCTTTCCGCCCTTC). The PCR product was digested by BamHI and ligated to the same restriction enzyme site in pL0011 (Malaria Research and Reference Reagent Resource Center, MR4 (http://www.malaria.mr4.org)). The plasmid was then digested by ApaI and integrated into the small subunit ribosomal DNA locus in P. berghei by single crossover recombination. The resultant transgenic parasite line, PbLuc expressed luciferase during all stages under the control of the elongation factor 1-α promoter.
Additional remarks selection procedure
top of page
Other details transgene
top of page
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative name
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
top of page
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c- or d-type unit)
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
top of page
Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren