Summary

RMgm-267
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: GFP (with ACP leader sequence for apicoplast targeting)
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)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c/d-type unit))
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 25 April 2009, 19:04
  *RMgm-267
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) : 19143588
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherR.R. Stanway, V.T. Heussler
Name Group/DepartmentBernhard Nocht Institute for Tropical Medicine
Name InstituteBernhard Nocht Institute for Tropical Medicine
CityHamburg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-267
Principal nameP. berghei-GFP(apico)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageUpon nuclear division, the single elongated apicoplast of the trophozoite stage becomes branched, with branches in many, but not all, cases appearing to extend towards parasite nuclei. In mature schizonts, each forming daughter parasite again contains an individual apicoplast.
Gametocyte/GameteIn female gametocytes, characterised by an elongated nucleus, the apicoplast exists as a single structure.
It is elongated, in many cases following the line of and appearing to physically contact the nucleus and in some, but not all, parasites it has one or two very short branches from the main structure. In mature male gametocytes no green-fluorescent organelle can be seen, but instead a weak cytoplasmic green fluorescence is observed.
Fertilization and ookineteOokinetes contain a single apicoplast, appearing similar in morphology to that of the female gametocyte.
OocystDuring oocyst development the apicoplast becomes increasingly branched, but appears to remain as a single organelle, forming a complex and seemingly random network. Mature oocysts (containing thousands of neatly aligned daughter sporozoites) displayed thousands of distinct “lines” of green fluorescence, whose pattern matched that of sporozoites in bright field images and also that of nuclear staining patterns.
SporozoiteIn sporozoites a single line of apicoplast fluorescence can be seen.
Liver stageThe apicoplast developed from a single elongated structure in the invading sporozoite, to become increasingly branched, forming a mesh of branches, in parallel with nuclear division. The branches do not appear to follow a defined pattern and do not appear to be necessarily associated with nuclei. During the cytomere, or meroblast stage of development, the apicoplast branches change from being smooth and curved to being angular and having a concertinaed morphology. On completion of schizogony, at which point the parasitophorous vacuole contains thousands of daughter merozoites, the apicoplast was seen to have divided into thousands of individual apicoplasts, seen as small distinct spots or lines of fluorescence.
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP that contains a targeting sequence for the apicoplast. This sequence is the leader sequence of the acyl carrier protein (ACP, PB000543.03.0). The gfp gene is under control of the constitutive eefia promoter. 

Protein (function)

Phenotype
The phenotype analyses indicate targeting of GFP to the apicoplast in all life cycle stages which allows for visualisation of the morphology and divison of the apicoplast and to analyse the effect of inhibitors on apicoplast division.

Additional information
Spinning disk microscopy confirmed that despite the highly branched nature of the apicoplast, the organelle remains as a single structure. 3D reconstructions of mature oocysts show very clearly the presence of thousands of individual apicoplasts, aligned in ring structures. Such images also show that the branched apicoplast structure has been divided, presumably into daughter sporozoites, with no residual undivided apicoplast remaining.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (with ACP leader sequence for apicoplast targeting)
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe pL0017 vector (obtained through the MR4 (MRA-786), allows constitutive expression of GFP under the control of the eef1a promotor, either by episomal expression or by integration of plasmid sequence into the cssu/dssu ribosomal gene locus of the parasite. The ACP (PB000543.03.0) leader sequence for apicoplast targeting was amplified from P. berghei blood stage genomic DNA using Phusion Taq DNA polymerase High Fidelity enzyme (Finnzyme) and the primers 5’- ATATGGATCCATGAAGATCATCTTACTTTCCATATTT-3’ and 5’- ATATGGATCCATAAAATGTACTATTCAAAGATGTTCTTG-3’ (restriction sites underlined). The fragment was introduced into the pL0017 vector upstream of the GFP coding sequence using the BamHI restriction site to create the plasmid pL0017-GFPapico. For transfection, plasmid DNA was linearised with ApaI and SacII.
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 / 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