Summary

RMgm-274
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP (mut3)
Promoter: Gene model: PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein
3'UTR: Gene model: PBANKA_0514900; Gene product: 28 kDa ookinete surface protein (Pbs21; P28)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (d-type unit))
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 1 May 2009, 10:11
  *RMgm-274
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) : 15186403
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherD. Vlachou, C.J. Janse, A.P. Waters, F.C. Kafatos
Name Group/DepartmentEuropean Molecular Biology Laboratory
Name InstituteEuropean Molecular Biology Laboratory
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-274
Principal name233cl1, cl2
Alternative namePbCTRPp.GFP
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteGFP expression starts a few hours after gametocyte activation, resulting in green-fluorescent late zygotes and ookinetes. GFP is present throughout the cytoplasm.
OocystGFP is present during oocyst development
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under control of the promoter of the ctrp gene (CSP and TRAP-related protein, CTRP))

Protein (function)
CTRP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP).  CTRP is located in the micronemes of ookinetes. CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells (see also mutants RMgm-140 and RMgm-141 that lack expression of CTRP).

Phenotype
The phenotype analyses indicate strong GFP expression during ookinete development and GFP is present during development of the oocysts. No GFP is detected in asexual blood stages and gametocytes. GFP expression starts a few hours after gametocyte activation. GFP expression was hardly detectable in activated female gametes that were defective in fertilisation, indicating that expression only occurs after fertilisation.

Additional information
This mutant has been used for real-time analysis of midgut invasion by the ookinete in the living mosquito as well as in explanted intact midguts. These studies permitted detailed analysis of parasite motile behaviour in the midgut and cell biological analysis of the invasion process.

Transcription of ctrp has been analysed by Northern analysis, showing detectable transcription 4h post gametocyte activation and increases in ookinetes to a maximum at 15–22 h.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (mut3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA 2.6 kb promoter fragment of the ctrp gene CTRPp) was amplified with the following PCR primers including PstI and BamHI sites, respectively, CTRPp-F: 5'-AAAACTGCAGTGAATATAATCGTACTATGAAAAC-3' and CTRPp-R: 5'-GCGGGATCCTTGTGTTTTGCTTTGTATTTAA-3'.
The ctrp promoter (CTRPp) was recloned as a PstI-BamHI fragment into a pUC19-derived plasmid, upstream of a 0.7 kb Pbs21 3'-UTR fragment, to give the pCTRPp.-.Sb plasmid vector. The gfp gene, was cloned as a BamHI fragment introduced into this plasmid between CTRPp and Pbs21 3'UTR. This expression cassette was cloned in a vector containing the d-ssu-rra gene as target sequence for homologous recombination by single cross-over integration of the construct.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0412900
Gene Model P. falciparum ortholog PF3D7_0315200
Gene productcircumsporozoite- and TRAP-related protein
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 15'-AAAACTGCAGTGAATATAATCGTACTAT GAAAAC-3'
Additional information primer 1CTRPp-F
Sequence Primer 25'-GCGGGATCCTTGTGTTTTGCTTTGTATTTAA-3'
Additional information primer 2CTRPp-R
3'-UTR
Gene Model of Parasite PBANKA_0514900
Gene product28 kDa ookinete surface protein
Gene product: Alternative namePbs21; P28
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 15'-AAT GCA CAG GTA CAG GAG AAA CTA AAT-3'
Additional information primer 1Pbs21-F
Sequence Primer 25'-CAC ACT CAT AAT GTT TTC CAG TCA ATT-3'
Additional information primer 2Pbs21-R
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (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