Summary

RMgm-268
Malaria parasiteP. berghei
Genotype
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;
Last modified: 26 December 2011, 18:12
  *RMgm-268
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) : 19380117
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherA. Combe, R. Menard
Name Group/DepartmentUnité de Biologie et Génétique du Paludisme
Name InstituteInstitut Pasteur
CityParis
CountryFrance
Name of the mutant parasite
RMgm numberRMgm-268
Principal nameTRAP/FlpL(-)
Alternative name
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
SporozoiteMidgut and salivary gland sporozoites express FlpL
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
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 (see below 'Protein-function'). 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 which was flanked by FRT sequences.

Protein (function)
In the Flp/FRT site-specific recombination (SSR) system of yeast, the Flp recombinase recognizes the 34 bp FRT sites and excises any DNA located between two directly oriented sites (referred to as the FRTed sequence). The activity of FlpL (Low activity) recombinase is greatly reduced at 37°C and above - temperatures in erythrocytic stages in the vertebrate host - and is maintained at 20°C –25°C, temperatures permissive for parasite development in the mosquito.

Phenotype
The phenotype analyses indicate that expression of FlpL is specific for midgut and salivary gland sporozoites. Expression of FlpL did not affect viability of infectivity of the different life cycle stages. By analysis of excision of the FRTed hdhfr selectable marker it was shown that FlpL was only active in the sporozoite stages and that the FlpL/FRT system in sporozoites was efficient in excision of FRTed DNA sequences from the genome.

Additional information
This mutant expressing FlpL can be used for introducing additional DNA sequences (genes) that are flanked by FRT sequences. Subsequently these sequences can be removed by mosquito transmission. This 'conditional mutagenesis' approach allows for the analysis of the effect of removal of genes essential for blood stage development. For an example of using this approach see mutant RMgm-270

Derivatives of the TRAP/FlpL(−) clones expressing bright GFP fluorescence via the constitutive HSP70 promoter are now available as parasite “receivers” of conditional alleles of genes of interest.

See also mutant UIS4/Flp(-)F (RMgm-269) for a mutant expressing Flp under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion.

Other mutants
RMgm-269: A mutant expressing Flp under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion. In addition it expresses GFP under the control of the constitutive hsp70 promoter.
RMgm-278: A mutant expressing Flp under the control of the uis4 promoter that is specifically switched on in sporozoites after salivary gland invasion. It does not contain a drug-selectable marker and does not express GFP.
RMgm-270: A 'conditional knock-out mutant of MSP-1', generated using the Flp/FRT system.


  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/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 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