Back to search resultsSummaryRMgm-1338
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Successful modification | The 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 | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei NK65 |
Name parent line/clone | P. berghei NK65 TRAP/FlpL(-) |
Other information parent line | TRAP/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). |
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The mutant parasite was generated by | |
Name PI/Researcher | Kalanon M; de Koning-Ward TF |
Name Group/Department | Molecular and Medical Research Unit, School of Medicine |
Name Institute | Deakin University |
City | Waurn Ponds, Victoria |
Country | Australia |
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Name of the mutant parasite | |
RMgm number | RMgm-1338 |
Principal name | FLP/EXP2-2A-FRT |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Reduced infectivity of sporozoites to mice |
Liver stage | Reduced infectivity of sporozoites to mice |
Additional remarks phenotype | Mutant/mutation 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. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1334300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1471100 | ||||||||||||||||||||||||||
Gene product | exported protein 2 | ||||||||||||||||||||||||||
Gene product: Alternative name | EXP2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The mutant contains a mutated exp2 gene with two FRT sites | ||||||||||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | The FRTed sequence of exp2 is removed in the sporozoite stage by the Flp/FRT system | ||||||||||||||||||||||||||
Additional remarks inducable system |
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 used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | EcoRI | ||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | See 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 | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | Flp/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 used | No | ||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The 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 | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||
Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||
Gene product: Alternative name | TRAP; SSP2; SSP-2 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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