Summary

RMgm-259
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP(gfp-mut3) fused to the HT-motif of the P. falciparum HRPII gene
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
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/d-type unit))
Phenotype Asexual bloodstage;
Last modified: 29 April 2009, 15:17
  *RMgm-259
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) : 18545649
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/ResearcherJ.J. MacKenzie, k. Haldar
Name Group/DepartmentDepartment of Pathology and Microbiology-Immunology
Name InstituteNorthwestern University, Feinberg School of Medicine
CityChicago, Illinois
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-259
Principal nameHRPIIminhis.HT.GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageHRPIIminhis.HT.GFP, green fluorescence was readily detected in the host erythrocyte. Green fluorescence was also detected in cytoplasm of the parasite.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP fused to a minitransgene containing the N-terminus with the HT-motif (host-targeting motif) of the histidine-rich protein II of P. falciparum (HRPIIminhis.HT.GFP) .
In the paper a second (control) mutant is described expressing GFP fused to a minitransgene containing an alanine-rich replacement HT-motif (HRPIIminhis.Δ.GFP).

Protein (function)
The host-targeting motif (HT-motif) has been identified as an 11-amino acid signal required for the secretion of proteins from the P. falciparum vacuole to the human erythrocyte ((Hiller, N.L. (2004) Science 306:1934-37). The truncated HRPII-proteins were previously used to define minimal soluble reporters needed to detect and establish HT-mediated export of GFP in P. falciparum  ( Lopez-Estrano C. et al (2003) Proc Natl Acad Sci U S A 100: 12402-12407).
The full-length PfHRPII protein (histidine rich protein) contains an N-terminal SS and HT motif, followed by a sequence rich in histidine-repeats that extends to the C terminus. In contrast, the minitransgene contains the HRPII N terminus with either the wildtype HT motif (HRPIIminhis.HT.GFP) or an alanine-rich replacement HT-motif (HRPIIminhis.Δ.GFP) followed by a single monomeric repeat of the histidine rich region fused to GFP.

Phenotype
HRPIIminhis.HT.GFP, green fluorescence was readily detected in the host erythrocyte. Green fluorescence was also detected in cytoplasm of the parasite, presumably because the constitutive eefia promoter is used to drive transgene expression.
In the mutant in which the HT-motif was replaced (HRPIIminhis.Δ.GFP) with an alanine-rich sequence the release of GFP into the erythrocyte cytoplasm was blocked. GFP was detected in the body of the parasite as well as tubular extensions connected to vesicular elements at the periphery of the erythrocyte.
These analyses indicate that the HT-motif can be utilized by P. berghei in exporting proteins to the erythrocyte cytoplasm and suggests that the machinery for HT-dependent export is conserved throughout the genus Plasmodium. They also indicate the presence of tubovesicular connections that extend between the vacuolar parasite and the periphery of the host erythrocyte, as detected in live cells.

Additional information

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP(gfp-mut3) fused to the HT-motif of the P. falciparum HRPII gene
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA previously described plasmid (pPbGFPCON or pL0016; www.mr4.org) was used for cloning of HRPIIminhis.HT.GFP (a truncated HRPII gene containing a minimal histidine sequence, host-targeting motif, and GFP tag) and HRPIIminhis.Δ.GFP (a truncated HRPII gene containing a minimal histidine sequence, an 11 amino acid alanine-rich replacement of the host-targeting motif, and GFP tag)(Lopez-Estrano C. et al (2003) Proc Natl Acad Sci U S A 100: 12402-12407). pL0016 contains a GFP expression cassette and the Toxoplasma gondii dihydrofolate reductase selectable marker (tgdhfr) for drug selection with perimethamine. HRPIIminhis.HT.GFP and HRPIIminhis.Δ.GFP was amplified from pBacII(HT-GFPmembmyc) and pBacII(Δ-GFPmembmyc), respectively, using the following primers: 5′ - ATAT GGATCC ATG GTT TCC TTC TCA AAA AAT AAA GTA TTA TCC – 3′ and 5′ - GGC CGG ATC CCT ATT TGT ATA GTT CAT CCA TGC CAT GTG TAA TCC C – 3′. pL0016 was digested with BamHI to remove GFP. PCR products for HRPIIminhis.WT.GFP and HRPIIminhis.Δ.GFP were digested with BamHI and ligated with pL0016. pL0016 (HTsol-GFP) and pL0016 (Δsol-GFP) were digested with ApaI and SacII.
The plasmid integrates into the c/d-type rrna gene unit by single cross-over integration (and not by double cross-over integration as mentioned in the paper!; see also RMgm5 for a mutant that is generated using pl0016 and expresses GFP).
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_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/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