Summary

RMgm-4570
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_1036400; Gene model (P.falciparum): PF3D7_1036400; Gene product: liver stage antigen 1 (LSA1)
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, 14:36
  *RMgm-4570
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-1314
Other information parent lineThis line expresses the PfLSA1 (accession no. PF3D7_1036400) 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 PfLSA1 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-4570
Principal name2374 cl1
Alternative namePbANKA-PfLSA1+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-1314. This line expresses the PfLSA1 (accession no. PF3D7_1036400) 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 Pbeef1α promoter. Both the PfLSA1 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-PfLSAP2+PbΔs1 GIMO; line 2374). 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-PfLSA1Pbuis4 parasites (line 2230 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_1036400
Gene Model P. falciparum ortholog PF3D7_1036400
Gene productliver stage antigen 1
Gene product: Alternative nameLSA1
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 modificationWe deleted the Pbs1 CDS and replaced it with a positive-negative selectable marker to create a Pbs1 deletion GIMO line (PbANKA-PfLSAP2+PbΔs1 GIMO; line 2374). 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-PfLSA1Pbuis4 parasites (line 2230 cl1) by using standard methods of transfection.
Additional remarks selection procedure
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 procedureWe deleted the Pbs1 CDS and replaced it with a positive-negative selectable marker to create a Pbs1 deletion GIMO line (PbANKA-PfLSAP2+PbΔs1 GIMO; line 2374). 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-PfLSA1Pbuis4 parasites (line 2230 cl1) by using standard methods of transfection.
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