RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4992
Malaria parasiteP. yoelii
Genotype
Transgene
 Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
3'UTR: Gene model: PBANKA_0403200; Gene product: circumsporozoite (CS) protein (CSP)
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230p)
Phenotype Sporozoite;
Last modified: 17 May 2021, 08:55
  *RMgm-4992
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) : 33750026
MR4 number
top of page
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-688
Other information parent lineGIMO-Py17X (RMgm-688; line 1923cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PY04774) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. yoelii 17XNL line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
top of page
The mutant parasite was generated by
Name PI/ResearcherHopp CS, Sinnis P
Name Group/DepartmentDepartment of Medicine, Division of Infectious Diseases
Name InstituteJohns Hopkins University School of Medicine
CityBaltimore
CountryUSA
top of page
Name of the mutant parasite
RMgm numberRMgm-4992
Principal namePymCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
top of page
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoitemCherry expression in sporozoites
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses mCherry under control of the csp regulatory sequences (3'and 5'-UTR). The mCherry expression cassette has been introduced into the neutral 230p locus (by GIMO transfection and negative selection).

Protein (function)

Phenotype
mCherry expression in sporozoites.

Additional information

Other mutants

  Transgene: Mutant parasite expressing a transgene
top of page
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
top of page
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe targeting plasmid pL1849‐mCherryCSP, containing sequences that would target the mcherry transgene to the P. yoelii 230p locus, and the mcherry coding sequence flanked by the P. berghei csp 5’UTR and the P. berghei dhfr 3’UTR was generated as follows: the 5’pbcsp‐mCherry‐3ʹpbdhfr cassette was amplified from plasmid pL0047 (RMgm database http://www.pberghei.eu), with primers pL0047‐PbCS‐mCherry‐F and pL0047‐PbCS‐mCherry‐R, which added XmaI sites for cloning into pL1849 (Lin et al, 2011), which contained the P. yoelii 230p locus (PY03857) 5'‐ and 3'‐targeting sequences. The resulting construct pL1849‐mCherryCSP was released from the plasmid through digestion with BstXI and ScaI and used for transfection into the P. yoelii line Py17XGIMO (line 1923cl1; Lin et al, 2011), according to standard procedures . The following day, negative selection with 1 mg/ml 5‐FC in drinking water was used to enrich for parasites that integrated the PbCSP‐mCherry‐3ʹpbdhfr cassette and excised the hdhfr::yfcu locus through double cross‐over homologous recombination.
Additional remarks selection procedure
top of page
Other details transgene
top of page
Promoter
Gene Model of Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
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_0403200
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
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 PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p; 230p
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
P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren