Summary

RMgm-4572
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1206800; Gene model (P.falciparum): PF3D7_1008600; Gene product: zinc finger (CCCH type) protein, putative (s1)
Transgene
Transgene Plasmodium: Gene model: PF3D7_1335900; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
Transgene not Plasmodium: GFP-Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 1 January 2019, 16:02
  *RMgm-4572
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-1317
Other information parent lineThis line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.
The mutant parasite was generated by
Name PI/ResearcherHalbroth BR, Spencer AJ, Janse CJ, Khan SM, Salman AM
Name Group/DepartmentThe Jenner Institute
Name InstituteUniversity of Oxford
CityOxford
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4572
Principal name2353 cl3
Alternative namePbANKA-PfTRAP+PbΔs1 GIMO line
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 stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
This GIMO mutant contains a hdfr/yfcu selection marker (SM) cassette in the neutral s1 (PBANKA_1206800) gene locus. The SM cassette has been introduced in the s1 locus of line RMgm-1317. This line expresses the PfTRAP (accession no. PF3D7_1335900) coding sequence (CDS) under the control of the Pbuis4 regulatory sequences and a fusion protein of GFP and firefly luciferase (LUC-IAV) under the control of the constitutive Pbeef1a promoter. Both the PfTRAP and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3.

Protein (function)

Phenotype
Normal development throughout the complete life cycle.

Additional information


Other mutant
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1206800
Gene Model P. falciparum ortholog PF3D7_1008600
Gene productzinc finger (CCCH type) protein, putative
Gene product: Alternative names1
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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWe deleted the Pbs1 CDS and replaced it with a positive-negative selectable marker to create a Pbs1 deletion GIMO line (PbANKA-PfTRAP+PbΔs1 GIMO; line 2353). In order to do this, we generated the pL1928 construct, based on the standard GIMO DNA construct pL0034. This construct contains a positive-negative (hdhfr::yfcu) SM cassette and was used to insert both the Pbs1 5′ and 3′ gene targeting regions (TRs). The linear pL1928 DNA construct was introduced into PbANKA-PfTRAPPbuis4 parasites (line 2281 cl1) by using standard methods of transfection.
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: Plasmodium
Gene Model of Parasite PF3D7_1335900
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2
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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationChimeric P. berghei parasites containing the P. falciparum gene of interest at the neutral 230p locus were generated following the ‘gene insertion/marker out’ (GIMO) technology as previously described, using the standard GIMO DNA construct pL0043. This construct contains 5′ and 3′ targeting sequences for the 230p locus as well as a multiple-cloning site for integration of transgene-expression cassettes. These constructs integrate by double crossover homologous recombination and replace the positive-negative selectable marker (SM) (human dihydrofolate reductase:: yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu)) cassette with the transgene-expression cassette. The expression cassette contained the transgene flanked by the 5′ and 3′ promoter and transcription terminator sequences of P. berghei UIS4, which were amplified from P. berghei ANKA wild-type (WT) genomic DNA. The coding sequence of the various P. falciparum genes were PCR amplified from P. falciparum genomic DNA (primer sequences are available upon request), apart from LSA1 and LSA3. Due to the large size of these open reading frames the coding sequence was amplified from plasmids used in the generation of the vaccine constructs (and hence codon optimized for expression in mammalian cells). In addition, a reporter-cassette containing GFP::luciferase52, driven by the constitutive P. berghei elongation factor 1 alpha (ef1α ) promoter, was also cloned into each transgene construct. The coding sequence and promoter region of all constructs was confirmed by sequencing. Linearized constructs were introduced into the GIMO parasites using standard methods of GIMO-transfection. Transfected parasites were selected in mice through addition of 5-fluorocytosine (5-FC) in drinking water, resulting in negative selection of parasites where the SM in the 230p locus was replaced by the expression/reporter-cassette.
Additional remarks selection procedureThis transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameETRAMP10.3
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_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameETRAMP10.3
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP-Luciferase
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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis transgenic parasite does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
Other details transgene
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
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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