RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4178
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: tdTomato
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: Not available; Gene product: Not available
Phenotype Oocyst; Sporozoite;
Last modified: 27 June 2017, 17:55
  *RMgm-4178
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) : 28617870
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/ResearcherRathnapala UL; McFadden GI
Name Group/DepartmentSchool of BioSciences
Name InstituteUniversity of Melbourne
CityParkville, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4178
Principal nameWT-tdTomato
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocysttdTomato expression in oocysts
SporozoitetdTomato expression in sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The parasite expresses tdTomato under control of the constitutive HSP70 promoter (and does not contain a drug selectable marker).

Protein (function)


Phenotype
No detailed analyses of tdTomato expression in different life cycle stages are provided

Additional information
The transgene expression cassette was introduced by double cross-over integration in a locus on P. berghei chromosome 6 between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220; see RMgm-757),

All parasite lines were tested in the complete life-cycle to confirm previous findings that HSP70 5’ driven expression of GFP, tdTomato and mCherry in this locus does not impair parasite development.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nametdTomato
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double 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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationLines expressing mCherry, GFP or tdTomato in the absence of a selectable marker were created in a two-step process. 5’ and 3’ flanks of the intergenic region of chromosome 6 (see RMgm-757) were generated by PCR using primers D331-D332 and D326-D327, respectively and cloned into the EcoRI/NotI (5’) and ApaI/AflII (3’) sites of pDONR-P2/P3 (Invitrogen) to create the plasmid pINT65’3’P2-P3. The hDHR/yfcu positive-negative selectable marker cassette from pL0034 (MRA-849) was cloned into pINT65’3’P2-P3 using XmaI/PstI. The construct was linearized with NotI/AscI and transfected into Pb ANKA parasites. Transfectants were cloned and correct integration confirmed by PCR.
GFP flanked by the HSP70 5’ and 3’ UTRs in pbc-GFP@hsp70 (a kind gift of Robert Menard, Institute Pasteur) was inserted into the SacI/XmaI sites of pBluescript (Agilent) to create pBSKHG. The mCherry sequence was cut out of pLChSKD with AvrII/BamHI and cloned into the NheI/BglII site of pBSKHG, replacing the GFP to create pBSKHCh. The tdTomato sequence was amplified and cloned into pL0006 as mCherry for pLChSKD, cut with AvrII/BamHI and cloned into the NheI/BglII site of pBSKHG to create pBSKHTm. pBSKHG, pBSKHTm and pBSKHCh were cut with SacI/XmaI to liberate the HSP705’-FP-HSP703’ cassette, which was inserted into pINT65’3’P2-P3 to create pINT65’3’HG, pINT65’3’HTm, and pINT65’3’HCh. These plasmids were linearized and transfected as above, with selection on 5-FC, and cloned. Correct gene insertion was confirmed by PCR. All parasite lines were tested in the complete life-cycle to confirm previous findings that HSP70 5’ driven expression of GFP, tdTomato and mCherry in this locus does not impair parasite development. The fidelity of all PCR amplified products was confirmed by sequencing.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 Not available
Gene productNot available
Gene product: Alternative name
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 Not available
Gene productNot available
Gene product: Alternative name
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