Summary

RMgm-273
Malaria parasiteP. berghei
Genotype
Transgene
Transgene Plasmodium: Gene model: PF3D7_1031000; Gene model (P.falciparum): PF3D7_1031000; Gene product: ookinete surface protein P25 (P25)
Promoter: Gene model: PBANKA_0515000; Gene model (P.falciparum): PF3D7_1031000; Gene product: ookinete surface protein P25 (Pbs25; P25)
3'UTR: Gene model: PBANKA_0515000; Gene product: 25 kDa ookinete surface antigen precursor (Pbs25; P25)
Replacement locus: Gene model: PBANKA_0515000; Gene product: 25 kDa ookinete surface antigen precursor (Pbs25; P25)
Phenotype Fertilization and ookinete; Oocyst;
Last modified: 31 July 2012, 17:55
  *RMgm-273
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) : 18316385
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherG. Mlambo, N. Kumar
Name Group/DepartmentMalaria Research Institute, Department of Molecular Microbiology and Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-273
Principal nameTrPfs25Pb
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 ookineteAnalysis of expression by IFA shows expression of Pfs25 in the ookinete stage. The protein is located on the surface of ookinetes.
OocystThe infectivity (number of oocysts per mosquito) and the rate of infectivity (percent infected mosquitoes) were comparable between these wild type and mutant parasites (60 ± 34 oocysts/midgut [wild type] versus 54 ± 10 [mutant]; 87.5% [wild type] versus 82.8% [mutant]infectivity; 80 and 64 mosquitoes were infected with wild type and mutant parasites, respectively
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses the ookinete surface protein P25 of P. falciparum (Pfs25). Pfs25 is under the control of the 5'-UTR and 3'-UTR regulatory regions p25 of P. berghei. The pfs25 gene is introduced in the p25-p28 locus of P. berghei, thereby disrupting both p25 and p28 which encode two major ookinete surface proteins of P. berghei.

Protein (function)
P25 is one of two major surface proteins (P25 and P28) of the plasma membrane of ookinetes. The proteins are conserved between different Plasmodium species. The proteins are characterized by a secretory N-terminal signal sequence followed by three or four epidermal growth factor (EGF) domains and a glycosylphosphatidylinositol (GPI) anchor. Synthesis of both proteins begins between 0.5 and 2 h after the formation of the female gametes, and the proteins are still present in young oocysts. The paralogous genes encoding P25 and P28 are located next to each other in the genome in a head to tail organization. Antibodies against P25 prevent the formation of oocysts in the mosquito and thereby block transmission of the parasite.

Phenotype
The phentype analyses of oocyst production indicate  that Pfs25 was able to functionally complement the combined functions of Pb25 and Pb28 without compromising parasite growth and infectivity to A. stephensi mosquitoes (see also RMgm-1, RMgm-2 and RMgm-3 for the phenotype of P. berghei mutants lacking expression of either P25 and P28 alone or lacking both proteins). Analysis of expression by IFA shows expression of Pfs25 in the  ookinete stage. The protein is located on the surface of ookinetes.

Additional information
Experiments described in the paper show that immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi mosquitoes Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These results show that the P. berghei mutant TrPfs25Pb can be used as a model for assessment of P. falciparum transmission-blocking vaccine.

Other mutants
RMgm-222, RMgm-223: P. berghei transgenic mutants expressing P25 of P. vivax.


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_1031000
Gene Model P. falciparum ortholog PF3D7_1031000
Gene productookinete surface protein P25
Gene product: Alternative nameP25
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe pfs25 gene was amplified using the primers Pfs25F sense, atcgatATGAATAAACTTTACAGTTTGTTTCT, nt +1 to +26; Pfs25R antisense, 5'-gaattcTTACATTATAAAAAAGCATACTC-3', nt +631 to +654.
The construct contained a 708bp sequence of the P28 gene of P. berghei for targeted integration of the construct into the P28 locus. This sequence was amplified using the following primers pb28-3' UTR (Pb28-3' UTR sense, 5'-ggatccTATATTCAATTGTTATCGC-3', nt 1 to 19 downstream from the pb28 stop codon; Pb28-3' UTR antisense, 5'-gcggccgcATTCGTATAAACACATTTC-3', nt 689 to 708 downstream from the pb28 stop codon). Recombination of the target sequences in the construct, the 5'-UTR of P25 and the 3'-UTR of P28, with the genomic P25-P28 locus results in disruption of the endogenous P25 and P28 genes of P. berghei, that are replaced with the P25 gene of P. falciparum.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0515000
Gene Model P. falciparum ortholog PF3D7_1031000
Gene productookinete surface protein P25
Gene product: Alternative namePbs25; P25
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'-ggtaccAGTTACATAAGCATTGTAAGAATTATTCA-3'
Additional information primer 1Pb25-5' UTR sense; nucleotides (nt) –649 to –620 upstream of pb25
Sequence Primer 25'-atcgatTTTTAAATAAATTTAAACGAATAAC-3'
Additional information primer 2Pb25-5' UTR antisense; nt –25 to –1 upstream of pb25
3'-UTR
Gene Model of Parasite PBANKA_0515000
Gene product25 kDa ookinete surface antigen precursor
Gene product: Alternative namePbs25; P25
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'-gaattcTTAAATAAACAAATATACCTGG-3'
Additional information primer 1Pb25-3' UTR sense; nt 1 to 22 downstream from the pb25 stop codon
Sequence Primer 25'-aagcttTTTCCTTATGCGCAG-3'
Additional information primer 2Pb25-3' UTR antisense; nt 585 to 600 downstream from the pb25 stop codon
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0515000
Gene product25 kDa ookinete surface antigen precursor
Gene product: Alternative namePbs25; P25
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 15'-ggtaccAGTTACATAAGCATTGTAAGAATTATTCA-3'
Additional information primer 1Pb25-5' UTR sense; nucleotides (nt) –649 to –620 upstream of pb25
Sequence Primer 25'-atcgatTTTTAAATAAATTTAAACGAATAAC-3'
Additional information primer 2Pb25-5' UTR antisense; nt –25 to –1 upstream of pb25
Sequence Primer 35'-ggatccTATATTCAATTGTTATCGC-3'
Additional information primer 3Pb28-3' UTR sense, nt 1 to 19 downstream from the pb28 stop codon
Sequence Primer 45'-gcggccgcATTCGTATAAACACATTTC-3'
Additional information primer 4Pb28-3' UTR antisense, nt 689 to 708 downstream from the pb28 stop codon