Summary

RMgm-285
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP fused to the amino terminal region of CSP (amino acids 1-74) with pexel motif mutations
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)
Phenotype Asexual bloodstage;
Last modified: 25 April 2011, 11:41
  *RMgm-285
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) : 17981117
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherA.P. Sing, V. Nussenzweig
Name Group/DepartmentDepartment of Pathology
Name InstituteNew York University School of Medicine
CityNew York
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-285
Principal nameCS-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageThe analysis of blood stages by fluorescence microscopy showed that the fusion CS-GFP gene entered the red blood cell cytoplasm. Mutation of the two putative pexel-/VTS motifs in the CS gene resulted in abortion of transport of the fusion gene into the cytoplasm of the red blood cell.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP fusion with the CS protein (CSP; PBANKA_040320; under control of the eef1α promoter). The CS amino terminal region (amino acids 1-74), which includes the signal sequence and putative pexel motifs, was cloned in-frame at the N-terminus of GFP followed by the 3’ P.berghei dhfr UTR for proper expression. The fusion cs-gfp gene was introduced by transient transfection using circular plasmids. In this study three additional mutants are described. Since P.berghei show two putative pexel motifs, three mutants were made. The first contains a mutation in the pexel1 motif, the second is mutated in pexel2, and the third has mutation in both motifs simultaneously. For pexel motif mutagenesis, the conserved arginine and leucine were mutated to alanine using the Quick change XL-II site directed mutagenesis kit (Stratagene, USA).
The cs fusion gene is under the control of the constitutive eef1a promoter, resulting in expression of CS in blood stages.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
The analysis of blood stages by fluorescence microscopy showed that the fusion CS-GFP gene when expressed in blood stages entered the red blood cell cytoplasm. Mutation of the two putative pexel/VTS motifs in the CS gene resulted in abortion of transport of the fusion gene into the cytoplasm of the red blood cell. These results indicate that the putative pexel/VTS motifs are involved in transport of the CS protein. See also mutant RMgm-286 in which the endogeneous cs gene is replaced with a cs gene with two mutated pexel/VTS motifs. This mutant shows a defective transport of CS into the cytoplasm/nucleus of the hepatocyte.

Additional information

Other mutants
RMgm-286: A mutant in which the endogeneous cs gene is replaced with a cs gene with two mutated pexel/VTS motifs.


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP fused to the amino terminal region of CSP (amino acids 1-74) with pexel motif mutations
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor transient transfection, pMD205 vector was used, which encodes P.berghei DHFR as pyrimethamine resistance marker. CS amino terminal region (amino acids 1-74) from P.berghei, which includes the signal sequence and putative pexel motifs, was cloned in-frame at the N-terminus of GFP followed by the 3’ P.berghei DHFR UTR for proper expression. The CS-GFP fusion protein expression was driven by the EF-1α promoter. Similarly, the mutant CS pexel motif fragments were cloned in the pMD205 vector. Since P.berghei show two putative pexel motifs, three mutants were made. The first contains a mutation in the pexel1 motif, the second is mutated in pexel2, and the third has mutation in both motifs simultaneously. For pexel motif mutagenesis, the conserved arginine and leucine were mutated to alanine using the Quick change XL-II site directed mutagenesis kit (Stratagene, USA).
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 / InsertionNot available
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