Summary

RMgm-1338
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1334300; Gene model (P.falciparum): PF3D7_1471100; Gene product: exported protein 2 (EXP2)
Details mutation: The mutant contains a mutated exp2 gene with two FRT sites
Details conditional mutagenesis: The FRTed sequence of exp2 is removed in the sporozoite stage by the Flp/FRT system
Transgene
Transgene not Plasmodium: FlpL recombinase of yeast (FlpL)
Promoter: Gene model: PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (TRAP; SSP2; SSP-2)
3'UTR: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Insertion locus: Gene model: PBANKA_1349800; Gene product: sporozoite surface protein 2 thrombospondin-related anonymous protein (sporozoite surface protein 2; SSP2; SSP-2)
Phenotype Sporozoite; Liver stage;
Last modified: 3 October 2015, 20:24
  *RMgm-1338
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 26347246
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone P. berghei NK65 TRAP/FlpL(-)
Other information parent lineTRAP/FlpL(-)(RMgm-268) is a P. berghei NK65 mutant that expresses the yeast Flpl recombinase under the control of the trap promoter. The mutant does not contain a drug-selectable marker (PubMed: PMID: 19380117).
The mutant parasite was generated by
Name PI/ResearcherKalanon M; de Koning-Ward TF
Name Group/DepartmentMolecular and Medical Research Unit, School of Medicine
Name InstituteDeakin University
CityWaurn Ponds, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-1338
Principal nameFLP/EXP2-2A-FRT
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteReduced infectivity of sporozoites to mice
Liver stageReduced infectivity of sporozoites to mice
Additional remarks phenotype

Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of EXP2. The mutant expresses the yeast Flp recombinase under the control of the trap promoter  and contains a mutated exp2 gene that contains two FRT sites. These  FRT sites are located in the 3'UTR region. This mutant has been generated by replacement of the endogenous exp2 gene by an 'FRTed' exp2 gene in mutant RMgm-268 that expresses Flp.

The exp2 locus locus is disrupted by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-268). Removal of the FRTed exp2 sequence has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the  exp2 sequence which was flanked by FRT sequences.

Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex.

Phenotype
Unsuccessful attempts to disrupt the exp2 gene using standard methods of gene disruption (see RMgm-916, RMgm-944, RMgm-1172) indicate an essential role of EXP2 during blood stage development.

In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to down-regulate EXP2 expression specifically in liver stages. Removal of the FRTed exp2 sequence has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of the  exp2 sequence which was flanked by FRT sequences.

The phenotype analyses indicate the EXP2 plays an important role during liver stage development. .

Additional information
See also mutants RMgm-1336, RMgm-1337, RMgm-1338.
Evidence is presented that despite the presence of EXP2 at the PVM throughout LS growth and despite KAHRPL-GFP  being translocated into the erythrocyte cytosol during the IDC, KAHRPL-GFP could not be actively  translocated across the PVM into the hepatocyte cytosol during LS development. In addition, evidence is presented that HSP101 is not expressed in liver stages.

Other mutants
See link for other EXP2 mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1334300
Gene Model P. falciparum ortholog PF3D7_1471100
Gene productexported protein 2
Gene product: Alternative nameEXP2
Details of the genetic modification
Short description of the mutationThe mutant contains a mutated exp2 gene with two FRT sites
Inducable system usedFlp/FRT
Short description of the conditional mutagenesisThe FRTed sequence of exp2 is removed in the sporozoite stage by the Flp/FRT system
Additional remarks inducable system
Click to view information
Click to hide information
A conditional mutagenesis approach was utilized that uses stage-specific expression of a temperature-sensitive variant of the yeast recombinase, flippase (FlpL) to catalyze the excision of DNA placed between two Flp Recognition Target sequences (FRT) (see RMgm-268). FlpL catalyzes site-specific DNA recombination at a temperature range of 25°-30°C.
The 3'UTR of exp2 was flanked by the two FRT-sites.
The plasmid was transfected into parasites that express FlpL under the control of the sporozoite-specific promoter of the Thrombospondin Related Anonymous Protein gene (TRAP/FlpL) (see RMgm-268), causing the excision of the FRT-flanked sequence specifically in sporozoites.
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 EcoRI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSee also mutant RMgm-1336, 1337, 1338.
To enable FLP/FRT conditional knockdown of EXP2 via deletion of its 3' UTR, the construct pEXP2-FRT was created. To achieve this, the EXP2 3' UTR was PCR amplified from P. berghei gDNA using primers MK73 and MK74, and inserted into the previously described p3' regFRT vector (Lacroix et al., 2011) at the HindIII and AvrII sites. A partial EXP2 CDS was then amplified with MK71 and MK72 and inserted into the vector at the HindIII and NotI sites,
ensuring that the linker region between the EXP2 stop codon and the FRT site was preserved (Lacroix et al., 2011). The final construct comprising the EXP2 CDS and the TRAP 3' UTR containing FRT sites was sequenced and linearised with HindIII prior to transfection. To enable conditional FLP/FRT mediated knockdown of EXP2, with an exported KAHRPL-GFP reporter and an mCherry reporter to ascertain expression of EXP2, the following fragments were inserted
sequentially into to the p3'regFRT vector: (i) the PbHSP70 promoter, amplified with DO241 and DO242 from P. berghei and inserted into HindIII/SphI; (ii) the N-terminal leader of the Knob-Associated Histidine Rich Protein conjugated to GFP (KAHRPL-GFP), amplified with DO243 and DO244 from pL0035-KAHRPL-GFP (Haase et al., 2013) and inserted into XhoI/SphI; (iii)
2A-mCherry, amplified with DO245 and DO246 and inserted into SphI/NheI; (iv) the P. berghei EXP2 3' UTR, amplified with DO251 and DO252 and inserted into MluI/SacII; and (v) the P. berghei EXP2 CDS, amplified with DO253 and DO254 and inserted into SacII/SphI. The final construct, termed pEXP2-2A-FRT was sequenced and linearised with SacII prior to transfection. Both pEXP2-FRT and pEXP2-2A-FRT constructs were independently transfected into PbNK65, PbNK65-(UIS4 5'/FLP) and PbNK65-(TRAP 5'/FLP).

The targeting constructs pEXP2-FRT or pEXP2-2A-FRT were designed such that upon integration into the exp2 locus, the EXP2 3' UTR would be replaced with the Thrombospondin Related Anonymous Protein (TRAP) 3' UTR and a selectable marker flanked by FRT-recombination sequences (Fig. 2A, B). The pEXP2-2A-FRT integration construct differs from pEXP2-FRT in
that the sequence encoding the EXP2 CDS is fused to the Foot and Mouth Disease Virus 2A peptide (2A) and mCherry, used as a reporter of EXP2 expression. Upon integration, the placement of the 2A peptide results in the production of a polycistronic mRNA transcript that results in separate polypeptides, with EXP2 tagged by 2A but not with mCherry (Straimer et al., 2012). Additionally, pEXP2-2A-FRT harbours an exported reporter cassette in which the N-terminal
leader of the Knob-Associated Histidine Rich Protein (KAHRPL) has been conjugated to GFP (Fig. 2B) and is under the transcriptional control of the strong constitutive promoter of HSP70.
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: not Plasmodium
Transgene nameFlpL recombinase of yeast (FlpL)
Details of the genetic modification
Inducable system usedFlp/FRT
Additional remarks inducable system The mutant expresses the yeast FlpL recombinase under the control of the promoter of trap. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences.
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences.
The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1349800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative nameTRAP; SSP2; SSP-2
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_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
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 PBANKA_1349800
Gene productsporozoite surface protein 2 thrombospondin-related anonymous protein
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2
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