Summary

RMgm-223
Malaria parasiteP. berghei
Genotype
Transgene
Transgene Plasmodium: Gene model: PVP01_0616100; Gene model (P.falciparum): PF3D7_1031000; Gene product: ookinete surface protein P25 (Pv25; 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 Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 2 November 2016, 10:57
  *RMgm-223
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) : 17049690
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherS. Ramjanee, C.J. Janse, R.E. Sinden
Name Group/DepartmentDivision of Cell and Molecular Biology
Name InstituteImperial College London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-223
Principal namePv25DR3
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteGametocytes transcribe the hybrid transgene pv25/pb25. However, the protein is not produced in this stage as the result of translational repression of the transcripts in the gametocyte stage. The protein is only expressed after induction of gamete formation in female gametes, zygotes and ookinetes.
Fertilization and ookineteExpression of Pv25/Pb25 (DR3) in female gametes, zygotes and ookinetes. The protein is located on the surface of ookinetes. The protein is evenly distributed over the ookinete surface.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a hybrid protein of the ookinete surface proteins P25 of P. vivax (Pv25) and P25 of P. berghei. Pv25/Pb25 is under the control of the 5'-UTR and 3'-UTR regulatory regions p25 of P. berghei. The pv25/pb25 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.
This mutant was generated using a construct that contains a 0.9 kb 5′ UTR of pb25, a truncated pv25 gene together with the GPI addition sequence of pb25, it's 3′ UTR and the entire (1.6 kb) intergenic region between pb25 and pb28 ORFs.

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.
Recombinant Histidine-tagged P25 of P. vivax (Pvs25) expressed in yeast (Pvs25H) has been tested in humans in phase I clinical trials and induces antibodies that blocked transmission significantly (up to 80% reduction in oocyst intensity and a 20–30% reduction in prevalence of infection).

Phenotype
Expression of Pv25/Pb25 (DR3) in female gametes, zygotes and ookinetes. The protein is located on the surface of ookinetes. The protein is evenly distributed over the ookinete surface.

Additional information
The transgenic parasites Pv25DR3 and Pv25DR (RMgm-122) have been used to develop assays to determine transmission blocking activity (TBA) of sera against P25 of P. vivax.. It was shown that oocyst development of the transgenic parasites is inhibited by monoclonal antibody against Pv25 with the same kinetics exhibited by wild type parasites when exposed to mouse monoclonal antibodies targeted to a paralogous protein P28. Human transmission-blocking sera from a clinical vaccine trial of Pv25 inhibited oocyst development of Pv25DR and Pv25DR3, whereas non-blocking sera did not. It was shown that  transmission-blocking activity can be determined in a simple assays of ookinete development in vitro, assays that obviate the need for mosquito colonies. These results demonstrate that transgenic rodent malarias expressing proteins from human Plasmodium species can be cheap, safe, and simple tools for testing TBA from sera.

Other mutants
RMgm-222: Another mutant expressing  P25 of P. vivax  (line Pv25DR). In this mutant the complete ORF of pv25 is expressed under the control of 5'-UTR and 3'-UTR regulatory sequences.  In this mutants the Pv25 protein is also located on the surface of ookinetes. In contrast to mutant Pv25DR3 described here, the protein is not evenly distributed over the ookinete surface, but is mainly located at one pole of the ookinete.
RMgm-273: A P. berghei transgenic mutant expressing P25 of P.falciparum.


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PVP01_0616100
Gene Model P. falciparum ortholog PF3D7_1031000
Gene productookinete surface protein P25
Gene product: Alternative namePv25; P25
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI, BamHI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationIn Pv25DR3 both endogenous p25 and p28 genes of P. berghei are disrupted and replaced with the hybrid gene pv25/pb25 under the control of the 5′ and 3′ UTRs of P. berghei pb25.
In P. berghei, pb25 and pb28 are located on chromosome 5 in a head-to-tail orientation separated by only 1.4 kb of intergenic sequence. The contiguity of these genes permits disruption of both using a single DNA vector.

The mutant was generated using a construct that contains a 0.9 kb 5′ UTR of pb25, a truncated pv25 gene together with the GPI addition sequence of pb25, it's 3′ UTR and the entire (1.6 kb) intergenic region between pb25 and pb28 ORFs. In addition the construct contains 5'-UTR sequences of pb25 and 3'-UTR sequences of pb28 as target sequences for integration into the genome by homologous recombination.


The truncated pvs25 gene lacking the GPI-addition sequence was cloned as an ApaI/XhoI fragment (Primers: sense 5′-GGGCCCATGAACTCCTACTACAGCCTC-3′, antisense 5′-CTCGAGTACATTTTTCTCTTTGTCGAAC).

A XhoI/ClaI fragment of the pb25 GPI addition sequence and pb25-pb28 intergenic region was cloned using the following primers: sense 5′-CTCGAGTGTATATCACATTCAATATATAG-3′, antisense 5′-ATCGATAAACTGTATTTAAAATTCAT-3′).
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 1GGTACCTTCATAGGGATATTTACATACTCC
Additional information primer 1Sense (KpnI)
Sequence Primer 2GGCCCTTTTAAATAAATTTAAACGAATAAC
Additional information primer 2Antisense (ApaI)
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 1CTCGAGTGTATATCACATTCAATATATAG
Additional information primer 1Sense (XhoI); pb25 GPI addition sequence and pb25-pb28 intergenic region
Sequence Primer 2ATCGATAAACTGTATTTAAAATTCAT
Additional information primer 2Antisense (ClaI); pb25 GPI addition sequence and pb25-pb28 intergenic region
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 1GGTACCTTCATAGGGATATTTACATACTCC
Additional information primer 1Sense (KpnI); Pb25 5'UTR
Sequence Primer 2GGCCCTTTTAAATAAATTTAAACGAATAAC
Additional information primer 2Sense (ApaI); Pb25 5'UTR
Sequence Primer 3GAATTCTATATTCAATTGTTATCGCATATTG
Additional information primer 3Sense (EcoRI); Pb28 3'UTR
Sequence Primer 4GGATCCGGCATAATCCTATTTTTAAGGAAAT
Additional information primer 4Antisense (BamHI); Pb28 3'UTR