Summary

RMgm-269
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: Flp recombinase of yeast (Flp)
Promoter: Gene model: PBANKA_0501200; Gene model (P.falciparum): Not available; Gene product: early transcribed membrane protein up-regulated in infective sporozoites
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_0501200; Gene product: early transcribed membrane protein up-regulated in infective sporozoites
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Insertion locus: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Sporozoite;
Last modified: 27 May 2012, 17:29
  *RMgm-269
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, 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-269
Principal nameUIS4/Flp(-)F
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
SporozoiteSalivary gland sporozoites express Flp
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome.
The hdhfr selectable marker used to introduce the Flp 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 Flp recombinase that resulted in the excision of the hdhfr gene which was flanked by FRT sequences.
The gfp gene has been introduced using a construct that contains a pyrimethamine resistant pbdhfr-ts gene after removal of the hdhfr gene as described above .

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 Flp recombinase is  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 Flp is specific for salivary gland sporozoites. Expression of Flp 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 Flp was only active in the sporozoite stages and that the Flp/FRT system in sporozoites was efficient in excision of FRTed DNA sequences from the genome. All life cycle stages express GFP.

Additional information
This mutant expressing Flp 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

The UIS4/Flp(−)F mutant is available as a parasite “receiver” of conditional alleles of genes of interest. This mutant contains the pbdhfr selectable marker. Therefore, introduction of additional mutations is dependent on a second drug selectable marker (hdhfr). See also mutant RMgm-278 for a mutant expressing Flp under the control of the uis4 promoter without expressing GFP and without a drug-selectable marker.

See also mutant TRAP/FlpL(-) RMgm-268 for a mutant expressing FlpL under the control of the trap promoter, resulting in expression of FlpL in both midgut- and salivary gland sporozoites.

Other mutants
RMgm-268: A mutant expressing FlpL under the control of the promoter of trap. It does not contain a drug-selectable marker.
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 nameFlp recombinase of yeast (Flp)
Details of the genetic modification
Inducable system usedFlp/FRT
Additional remarks inducable system The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. 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 Flp 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 gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker 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 Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. In the resulting mutant (UIS4/Flp(-), by integrating at the DHFR-TS locus a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0501200
Gene Model P. falciparum ortholog Not available
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
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 15'-GCGCCCCGGGTGTGATAGTGTAGATTTTTTTGTTTGACCATTG-3'
Additional information primer 1
Sequence Primer 25'- GCGCCCATGGTTTATTCAGACGTAATAATTATGTGCTGAAAGG-3'
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_0501200
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative name
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe flpl gene was cloned between 1.5 kb of 5′ regulatory sequence of uis4 and 0.6 kb of 3′ regulatory sequence of trap. The plasmid was linearized in the 5′ uis4 regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous uis4 gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. The mutant expresses the yeast Flp recombinase under the control of the promoter of uis4. The mutant does not contain the hdhfr selectable marker that has been used to introduce the flp gene. This marker 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 Flp recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. In the resulting mutant (UIS4/Flp(-), by integrating at the DHFR-TS locus a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene.

To make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant pbdhfr-ts gene
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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