Summary

RMgm-234
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1006200; Gene model (P.falciparum): PF3D7_0408600; Gene product: sporozoite invasion-associated protein 1 (Sporozoite Invasion-Associated Protein-1; SIAP-1; SIAP-1/ag17/S5)
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_1006200; Gene product: sporozoite invasion-associated protein 1 (Sporozoite Invasion-Associated Protein-1; SIAP-1; SIAP-1/ag17/S5)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 24 April 2009, 09:03
  *RMgm-234
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) : 19181869
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/ResearcherS. Engelmann, K. Matuschewski
Name Group/DepartmentDepartment of Parasitology
Name InstituteHeidelberg University School of Medicine
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-234
Principal namesiap-1(-)/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
OocystNormal numbers of oocyst and oocyst-derived sporozoites are formed. In a significant fraction of mature oocysts the sporozoites did not egress from the oocysts resulting in reduced numbers of sporozoites in the hemocoel.
SporozoiteNormal numbers of oocyst and oocyst-derived sporozoites are formed. In a significant fraction of mature oocysts the sporozoites did not egress from the oocysts resulting in reduced numbers of sporozoites in the hemocoel. No sporozoites were detected in the salivary glands. Sporozoites show infrequent, non-productive motility.
Liver stageMice/rats could not be infected by the bite of mosquitoes infected with the mutant.
Only by intravenous injections of high numbers of sporozoites (50.000), blood stage infections developed in mice.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SIAP-1 (Sporozoite Invasion-Associated Protein-1) and expresses GFP under the control of the csp promoter.

Protein (function)
SIAP-1 orthologs have been found exclusively in apicomplexan hemoprotozoa, parasites that are transmitted by arthropod vectors, e.g., Plasmodium, Babesia, and Theileria species (and is absent in, for example,  Toxoplasma and Cryptosporidium).  SIAP1 was first discovered in a systematic screen for P. falciparum sporozoite antigens recognized by sera from individuals that were immunized with irradiated sporozoites and termed antigen 17 (ag17). Subsequently, the P. yoelii ortholog was isolated in a suppression subtractive hybridization screen for genes that are specifically upregulated in sporozoites (S5) compared to blood stages. Analyses of a mutant expressing (C-terminal) mCherry-tagged form of SIAP-1 (RMgm-232). showed specific expression of SIAP-1 in sporozoites and liver stages.

Phenotype
The phenotype analyses indicate a partial defect in the egress of sporozoites  from oocysts and indicate an additional effect on invasion of salivary glands. Sporozoites did not show gliding motility and a  complete block of sporozoite transmission from highly infected mosquitoes was observed.

See also the phenotype analyses of mutant RMgm-109 which indicate that sporozoites lacking expression of SIAP-1are not affected in infectivity.

Additional information
See also Vignali, M. et al. (2009, Genome Biology, 10:216) for a discussion on the slightly different phenotypes reported for mutant RMgm-109 and RMgm-233, RMgm-234.

Other mutants
RMgm-109: A mutant lacking expression SIAP-1
RMgm-232: A mutant expressing (C-terminal) mCherry-tagged form of SIAP-1
RMgm-233: A mutant lacking expression SIAP-1
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1006200
Gene Model P. falciparum ortholog PF3D7_0408600
Gene productsporozoite invasion-associated protein 1
Gene product: Alternative nameSporozoite Invasion-Associated Protein-1; SIAP-1; SIAP-1/ag17/S5
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
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe wild-type siap-1 locus is targeted with a construct containing the 5´and 3´ UTR of Pbsiap-1, the positive selection marker Tgdhfr-ts, and the gfp coding sequence under control of the csp promoter region. After double cross-over homologous recombination, the siap-1 open reading frame is substituted by the selection marker and the gfp cassette.
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 1TCCCCGCGGAGTTAACTCGAAAAAAATAAGGACG
Additional information primer 1SIAP-1_REP1 (SacII)
Sequence Primer 2ATAAGAATGCGGCCGCTTTTAAGATATACCCCTTAATGAGAGC
Additional information primer 2SIAP-1_REP2 (NotI)
Sequence Primer 3CCCAAGCTTGATGTATGTATAATACCGAAAATTGG
Additional information primer 3SIAP-1_REP3 (HindIII)
Sequence Primer 4CGGGGTACCCGCCTTATTTAAGGGATAAAGAATGGG
Additional information primer 4SIAP-1_REP4 (KpnI)
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 SacII/KpnI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe wild-type siap-1 locus is targeted with a construct containing the 5´and 3´ UTR of Pbsiap-1, the positive selection marker Tgdhfr-ts, and the gfp coding sequence under control of the csp promoter region. After double cross-over homologous recombination, the siap-1 open reading frame is substituted by the selection marker and the gfp cassette.
Additional remarks selection procedure
Other details transgene
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
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_1006200
Gene productsporozoite invasion-associated protein 1
Gene product: Alternative nameSporozoite Invasion-Associated Protein-1; SIAP-1; SIAP-1/ag17/S5
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 1TCCCCGCGGAGTTAACTCGAAAAAAATAAGGACG
Additional information primer 1SIAP-1_REP1 (SacII)
Sequence Primer 2ATAAGAATGCGGCCGCTTTTAAGATATACCCCTTAATGAGAGC
Additional information primer 2SIAP-1_REP2 (NotI)
Sequence Primer 3CCCAAGCTTGATGTATGTATAATACCGAAAATTGG
Additional information primer 3SIAP-1_REP3 (HindIII)
Sequence Primer 4CGGGGTACCCGCCTTATTTAAGGGATAAAGAATGGG
Additional information primer 4SIAP-1_REP4 (KpnI)