RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4520
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1359600; Gene model (P.falciparum): PF3D7_1346700; Gene product: 6-cysteine protein | transmission blocking target antigen precursor (P48/45)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_1359600; Gene product: 6-cysteine protein | transmission blocking target antigen precursor (P48/45)
Phenotype Oocyst;
Last modified: 11 September 2018, 17:49
  *RMgm-4520
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) : 30181253
MR4 number
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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
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The mutant parasite was generated by
Name PI/ResearcherCao Y, Kumar N
Name Group/DepartmentDepartment of Tropical Medicine, School of Public Health and Tropical Medicine
Name InstituteTulane University
CityNew Orleans
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-4520
Principal namePbs48/45 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
OocystNo oocyst formation in A. stephensi mosquitoes
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of P45/48 and expresses GFP under control of the constitutive eef1a promoter.

Protein (function)
The P48/45 protein (PF13_0247) 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 (Thompson et al., 2001). 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.
P48/45 is a surface protein of both male and female gametes and is considered as a major candidate antigen for development of a transmission blocking vaccine.
Phenotype analyses of P. berghei mutants lacking expression of P45/48 (RMgm-15) demonstrate that P48/45 plays an important role in fertilization (male fertility factor). Motile males fail to attach to and penetrate female gametes. Mutant female gametes are fertile as has been shown by cross-fertilisation with wild type male gametes.

Phenotype
The phenotype has not been analysed in much detail. No oocyst formation in A. stephensi mosquitoes. This mutant has been used as a control for P. berghei mutants expressing P45/48 of P. vivax in place of P. berghei P45/48 (RMgm-4521, RMgm-4522).

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1359600
Gene Model P. falciparum ortholog PF3D7_1346700
Gene product6-cysteine protein | transmission blocking target antigen precursor
Gene product: Alternative nameP48/45
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid pL0006 (MRA-775; BEI Resources, Inc.) was modified by replacing pbef 5' UTR-hdhfr with a cassette consisting of pbef 5' UTR-hdhfr-gfp. Using pL0006 as the template, pbef 5' UTR and hdhfr regions were amplified using primers 858/859 and 860/861, respectively. PCR-amplified pbef 5' UTR was cloned by TA cloning in pCR2.1-TOPO vector (Invitrogen), followed by insertion of PCR-amplified hdhfr into the MluI and SalI sites. Next, the PCR-amplified gfp gene (amplified using primers 862/863 from plasmid pL0021 [MRA-790; BEI Resources, Inc.]) was cloned into pCR2.1-TOPO vector downstream of hdhfr using SalI and BamHI sites. Forward primer 862, used for amplification, also contained 5 codons to insert a linker of five alanine residues to separate hDHFR and GFP polypeptide domains within the fusion protein. The gene cassette containing pbef 5' UTR and a fused hdhfr-gfp gene in the pCR2.1-TOPO vector was used to replace the fragment of pbef 5' UTR-hdhfr in pL0006 using PstI and BamHI to obtain plasmid pL0006-gfp.

To generate the knockout (KO) plasmid for homologous recombination by double crossover, pbs48/45 5' UTR and pbs48/45 3= UTR fragments were obtained by PCR amplification using genomic DNA as the template and primers 864/865 and 870/886, respectively. The pbs48/45 5' UTR fragment was inserted into plasmid pL0006-gfp using ApaI and SacII sites (Fig. S1). Likewise, the pbs48/45 3' UTR fragment was inserted in the Xhol and NotI sites. In addition, the pbdhfr-ts 3' UTR (pbdt 3' UTR) gene was amplified from plasmid pL0006 using primers 884/885 and inserted between the BglII and PstI restriction sites to obtain the KO plasmid for pbs48/45 knockout in P. berghei parasites.
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid pL0006 (MRA-775; BEI Resources, Inc.) was modified by replacing pbef 5' UTR-hdhfr with a cassette consisting of pbef 5' UTR-hdhfr-gfp. Using pL0006 as the template, pbef 5' UTR and hdhfr regions were amplified using primers 858/859 and 860/861, respectively. PCR-amplified pbef 5' UTR was cloned by TA cloning in pCR2.1-TOPO vector (Invitrogen), followed by insertion of PCR-amplified hdhfr into the MluI and SalI sites. Next, the PCR-amplified gfp gene (amplified using primers 862/863 from plasmid pL0021 [MRA-790; BEI Resources, Inc.]) was cloned into pCR2.1-TOPO vector downstream of hdhfr using SalI and BamHI sites. Forward primer 862, used for amplification, also contained 5 codons to insert a linker of five alanine residues to separate hDHFR and GFP polypeptide domains within the fusion protein. The gene cassette containing pbef 5' UTR and a fused hdhfr-gfp gene in the pCR2.1-TOPO vector was used to replace the fragment of pbef 5' UTR-hdhfr in pL0006 using PstI and BamHI to obtain plasmid pL0006-gfp.

To generate the knockout (KO) plasmid for homologous recombination by double crossover, pbs48/45 5' UTR and pbs48/45 3= UTR fragments were obtained by PCR amplification using genomic DNA as the template and primers 864/865 and 870/886, respectively. The pbs48/45 5' UTR fragment was inserted into plasmid pL0006-gfp using ApaI and SacII sites (Fig. S1). Likewise, the pbs48/45 3' UTR fragment was inserted in the Xhol and NotI sites. In addition, the pbdhfr-ts 3' UTR (pbdt 3' UTR) gene was amplified from plasmid pL0006 using primers 884/885 and inserted between the BglII and PstI restriction sites to obtain the KO plasmid for pbs48/45 knockout in P. berghei parasites.
Additional remarks selection procedure
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Other details transgene
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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
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3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
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_1359600
Gene product6-cysteine protein | transmission blocking target antigen precursor
Gene product: Alternative nameP48/45
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren