RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4067
Malaria parasiteP. berghei
Genotype
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; UIS4)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3; UIS4)
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)
Transgene
Transgene Plasmodium: Gene model: PF3D7_1302200; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (ETRAMP13; UIS3)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3; UIS4)
3'UTR: Gene model: PBANKA_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (ETRAMP10.3; UIS4)
Replacement locus: Gene model: PBANKA_1206800; Gene product: zinc finger (CCCH type) protein, putative
Phenotype Sporozoite;
Last modified: 9 January 2017, 18:32
  *RMgm-4067
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28031267
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone GIMO-PbANKA (RMgm-687)
Other information parent lineGIMO-PbANKA (RMgm-687) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
The mutant parasite was generated by
Name PI/ResearcherLongley RJ, Salman AM, HillAV, Janse CJ, Khan SM, Spencer AJ
Name Group/DepartmentThe Jenner Institute
Name InstituteUniversity of Oxford
CityOxford
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4067
Principal name2395cl1
Alternative namePfTRAP+PfUIS3@Pbuis4 DAG
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
SporozoiteP. falciparum TRAP and UIS3 expression in sporozoites
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses P. falciparum TRAP and UIS3 under control of the sporozoite/liver stage promoter uis4 and expresses the reporter fusion protein GFP-Luciferase under control of the constitutive eef1a promoter.

The TRAP-UIS4 expression cassette has been introduced into the silent 230p locus (see mutant RMgm-1317). In this mutant the UIS3-UIS4 expression cassette has been introduced by GIMO transfection into the silent s1 locus (PBANKA_1206800).

The mutant does not express a drug-selectable marker

Protein (function)

Phenotype
Both transgenes (P. falciparum TRAP and UIS3) are expressed in sporozoites. The mutant shows normal development throughout the life cycle.

Additional information
The mutant has been used to evaluate vaccines in vivo targetting P. falciparum TRAP and UIS3

Other mutant
 


  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 modificationGeneration of Pb-Pf double chimeric parasites expressing both PfUIS3 and PfTRAP. To generate the Double Additional Gene (DAGs) chimeric parasite PbANKA-PfTRAP+PfUIS3@Pbuis4 DAG (line 2395cl1) we used the previously generated single additional gene (SAG) PbANKA-PfTRAPPbuis4 line (line 2281cl1) as the background parent line. This line expresses the PfTRAP (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 constitutive Pbeef1a promoter. This transgenic parasite is selectable marker (SM) free. Both the trap and Luc-gfp expression cassettes are integrated into the neutral 230p locus in chromosome 3. In this line the PfUIS3 CDS (PfUIS3; PF3D7_1302200) was stably integrated into a neutral s1 gene locus (Pbs1; PBANKA_120680), through double cross-over recombination using a 2-step GIMO transfection protocol. In the first step we deleted the Pbs1 CDS and replaced it with the positive negative selectable marker, to create a Pbs1 deletion GIMO line (PbANKA-PfTRAP+PbΔs1 GIMO; line 2353cl2). In order to do this we generated the pL1928 construct that is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative (hdhfr::yfcu) SM cassette, and was used to insert both the Pbs1 5’ and 3’ gene targeting regions (TR). The linear pL1928 DNA construct was introduced into PbANKA-PfTRAPPbuis4 parasite (2281cl1) using standard methods of transfection. Transfected parasites were selected in mice through addition of pyrimethamine in the drinking water. Transfected parasites were cloned by limiting dilution, resulting in the PbANKA-PfTRAP+PbΔs1 GIMO; line 2353cl2. Correct deletion of the Pbs1 CDS and its replacement with the SM cassette were confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel separated chromosomes as described.

In the second step we replaced the positive-negative SM in the genome of the PbANKA PfTRAP+PbΔs1 line (line 2353cl2) with the PfUIS3 CDS by GIMO transfection to create the Pb DAGs chimeric line expressing PfTRAP and PfUIS3. This was achieved by modifying the construct used in the first step (pL1928); specifically, the hdfhr::yfcu SM cassette was removed and replaced with PfUIS3 CDS expression cassette, generating plasmid pL2043. The expression cassette in pL2043 contained the PfUIS3 CDS flanked by the 5’ and 3’ regulatory sequences of Pbuis4, which were amplified from Pb ANKA WT genomic DNA. The PfUIS3 CDS was amplified from genomic DNA of the NF54 strain of Pf. The pL2043 construct was sequenced to ensure there were no mutations in the PfUIS3 CDS. The construct was linearized using HindIII restriction enzymes outside of the 5’ and 3’ Pbs1 TRs before transfection. The construct was used to transfect parasites of the PbANKA-PfTRAP+PbΔs1 GIMO (line 2353cl2) using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. This resulted in selection of chimeric parasites where the hdhfr::yfcu SM in the Pbs1 locus of PbANKA-PfTRAP+PbΔs1 GIMO line was replaced by the PfUIS3 CDS expression cassette. Selected chimeric parasites were cloned by limiting dilution.
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; UIS4
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; UIS4
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 procedure
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PF3D7_1302200
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameETRAMP13; UIS3
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 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; UIS4
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; UIS4
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_1206800
Gene productzinc finger (CCCH type) protein, putative
Gene product: Alternative name
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