Summary

RMgm-406
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: PiggyBac transposase
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c/d-type unit))
Phenotype Asexual bloodstage;
Last modified: 23 March 2011, 15:27
  *RMgm-406
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) : 21418605
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/ResearcherJ. Fonager; C.J. Janse; A.P. Waters
Name Group/DepartmentLeiden Malara Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-406
Principal name1042cl1
Alternative nameTPSama1
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageBlood stages express piggyBac transposase under the control of the schizont specific ama1 promoter
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses piggyBac transposase under the control of the schizont specific ama1 promoter. The mutant contains 2 copies of the piggyBac transposase integrated into the c/d-ssu-rrna gene (integrated by single cross-over recombination).

In the paper describing this mutant two different approaches were used to obtain insertion of piggyBac elements into the genome of P. berghei. In the first approach parasites were ‘co-transfected’ with both piggyBac donor plasmid (pL1302) and the helper plasmid (pL1301). The helper plasmid contains the transposase under the control of the constitutive eef1a promoter and this plasmid does not contain a drug-selection cassette (see Figure). Since plasmids are not retained in parasites during asexual growth without drug-selection the helper plasmid is ‘transiently transfected’ and will be lost from the parasites during blood stage growth.

In the second approach the donor plasmid was transfected into transgenic parasites described here that contain the transposase gene stably integrated into the c/d-ssu-rrna gene locus. This transgenic line, TPSama1, contain the T. gondii dhfr/ts as a selectable marker and transposase under the control of the schizont specific ama-1 promoter (see Figure).

Using both methods a large number of piggyBac inserts into the P. berghei genome have been identified. All genes with piggyBac inserts in the ORFor within the 5’UTR region (500 bp from the start of the ORF) have been deposited in this database.

The donor plasmid contains the 5’and 3’ inverted terminal repeats of the piggyBac element. which are the minimal cis elements necessary for piggyBac mobilization. Both inverted repeat sequences consist of a terminal 13 bp and internal 19 bp perfect inverted repeat that are separated by a 3 bp (5′ITR) or a 31 bp (3′ITR) spacer. In the donor plasmid the two ITR sequences are located on both sides of a drug-selectable marker cassette and a gfp expression cassette that lacks a promoter region (see Figure). The target site for piggyBac insertion is TTAA and it moves by precise insertion and excision mechanisms. Transfection of the donor plasmid would therefore result in insertion of both the drug-selectable marker cassette and the gfp-expression site without leaving a footprint at an insertion site. Insertion of the drug selectable marker, the human dhfr gene, allows for selection of parasites containing the inserts using pyrimethamine or WR99210.

 


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namePiggyBac transposase
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid single 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 parasitetgdhfr
Promoter of the selectable markerama-1
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutant contains 2 copies of the piggyBac transposase integrated into the c/d-ssu-rrna gene.

The mutant has been generated using a construct that integrates by a single cross-over integration event (insertion vector). The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative name
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 / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c/d-type unit)
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