Summary

RMgm-592
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1002200; Gene model (P.falciparum): PF3D7_0404500; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1002200; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
Insertion locus: Gene model: PBANKA_1002200; Gene product: 6-cysteine protein (P36p; Pb36p; P52)
Phenotype Liver stage;
Last modified: 24 December 2010, 17:46
  *RMgm-592
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20921402
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/ResearcherP. Gueirard; R. Amino
Name Group/DepartmentUnité de Biologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-592
Principal nameP36p-G; ΔP36p-GFP
Alternative name
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageBased on phenotype analyses of mutants RMgm-40 and RMgm-44 that lack expression of P36p/P52 (In this study these parasites have been used to analyse sporozoites in the skin of mice):
Gliding motility, hepatocyte traversal and hepatocyte invasion in vitro (HepG2) of mutant sporozoites is similar to wild type sporozoites. Liver stage development is strongly impaired and parasites do not develop into the schizont stage and most invaded parasites cannot be detected anymore at 24 hours after infection. Inside the hepatocytes the formation of a parasitophourous vacuole is not detected at 15 and 24h after infections as shown by staining with antibodies against the parasithophorous vacuole membrane protein PbExp1 (HEP17).
Infection of mice (C57BL/6) by bite of infected mosquitoes did not result in blood stage infection. Only intravenous inoculation of very high numbers of sporozoites (50.000) resulted in blood stage infection in a very low percentage of the mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the P36p/P52 protein and expresses the reporter protein GFP under the control of the hsp70 promoter.

Protein (function)
P36p/P52 is a member of a small family of proteins, the 6-cysteine (cys) family of (surface) proteins (Thompson J. et al., Mol. Biochem. Parasitol. (2001)118, 147-54). The proteins are characterized by domains of roughly 120 amino acids in size that contain six positionally conserved cysteines (6-cys). Although some species of Plasmodium (may) contain unique members of the 6-cys family, ten members have been identified that are conserved both in structure as well as in genome organization throughout the genus. Some of the conserved 6-cys proteins are encoded by genes that form paralogous gene-pairs which are closely linked in the genome separated by less then 2 kb of intergenic region. Most members have a GPI anchor and are predicted membrane surface proteins whereas others appear to be secreted and most members are expressed in a discrete stage-specific manner in gametocytes, sporozoites or merozoites.
A paralogue of the p36p gene, p36 (PB000892.00.0; pbs36), is located in the genome next to p36p (in tandem).

Phenotype
For the phenotype of parasites lacking expression of P36p see the description of the mutants RMgm-40 and RMgm-44. In this study these parasites have been used to analyse sporozoites in the skin of mice.

Other mutants

A P. berghei mutant has been generated that lacks expression of this protein without the expression of the reporter protein GFP (RMgm-40).
An independent P. berghei mutant has been generated that lacks expression of this protein (RMgm-44).
A P. berghei mutant (RMgm-42) has been generated that lacks the expression of not only this protein but also its paralogue P36 (PB000892.00.0; pbs36).
A P. yoelii mutant (RMgm-43) has been generated that lacks the expression of not only this protein but also its paralogue P36 (PY01341; p36).
In P. falciparum a mutant has been generated lacking this protein (PFD0215c; van Schaijk et al., (2008) PloS ONE 3(10):e3549). The 'arrested' phenotype of liver stage of this P. falciparum mutant in primary human hepotocytes is similar to the phenotype of the P. berghei mutant.

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1002200
Gene Model P. falciparum ortholog PF3D7_0404500
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption Other than figure S1 showing a schematic of the construct, the paper provides very little information regarding target regions and primers used to amplify the target regions
Selectable marker used to select the mutant parasitegfp (FACS)
Promoter of the selectable markerhsp70
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationP36p gene disrupted P. berghei parasites were generated by double- crossing-over homologous recombination using GFP gene as a selectable marker. After transfection of linearized plasmid into schizonts infected erythrocytes, transgenic blood-stage parasites were selected by cell sorting using GFP signal. The mutant does not contain a drug-selectable marker
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: 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
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 parasitegfp (FACS)
Promoter of the selectable markerhsp70
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP transgene was introduced using a double cross-over strategy. Figure S1 shows the target regions being adjacent. Therefore, GFP was inserted into the P36p locus without replacement of sequence.

Other than figure S1, the paper provides very little information regarding target regions and primers used to amplify these regions
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 PBANKA_1002200
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
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 PBANKA_1002200
Gene product6-cysteine protein
Gene product: Alternative nameP36p; Pb36p; P52
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