Summary

RMgm-270
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0831000; Gene model (P.falciparum): PF3D7_0930300; Gene product: merozoite surface protein 1 (MSP-1, MSP1)
Details mutation: The mutant contains a FRTed 3'UTR of the msp-1 locus
Details conditional mutagenesis: The 3'UTR of msp1 is removed in the sporozoite stage by the Flp/FRT system.
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; Liver stage;
Last modified: 22 December 2009, 18:49
  *RMgm-270
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, 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-270
Principal nameMSP1/Cond
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
SporozoitePCR and RTqPCR analysis of genomic DNA of the mutant sporozoites showed high levels (>95%) of SSR (site-specific recombination) in salivary gland sporozoites, which resulted in the removal of the FRTed 3'UTR of MSP-1.
The mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites.
Liver stageThe mutant sporozoites showed an infectivity to HepG2 cells that was comparable to wild type parasites and produced livers stages at 24, 48, 63 and 71 hour after incubation of mutant sporozoites with HepG2 cells that were comparable in number to that of wild type sporozoites.
The liver stages of the mutant yielded on average 30-fold fewer merozoites than wild type liver stages (see also 'Additional remarks phenotype').
Additional remarks phenotype

Mutant/mutation
The mutant is a 'Flp/FRT conditional knock-out mutant' of MSP-1 (merozoite surface protein 1, precursor). The mutant expresses the yeast Flp recombinase under the control of the uis4 promoter  and contains a FRTed 3'UTR of the msp-1 locus. In addition it expresses GFP under the control of the hsp70 promoter. The gfp gene is stably integrated into the genome. This mutant has been generated by replacement of the endogenous msp-1 gene by an msp-1 gene with a FRTed 3'UTR in the mutant RMgm-269 that expresses Flp and GFP.
The 3'UTR of msp-1 locus is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269). Removal of the FRTed 3'UTR of msp-1  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 3'UTR of msp1 which was flanked by FRT sequences.

Protein (function)
Merozoite attachment to the erythrocyte surface is mediated by a family of proteins called merozoite surface proteins (MSPs). MSP-1, is a glycosylphosphatidylinositol (GPI)-anchored protein that covers the entire surface of free merozoites. Antibody inhibition experiments and and molecular genetic studies have shown that MSP-1 is essential for merozoite adhesion to erythrocytes.  In addition, expression of MSP-1 is switched on during the first half and peaks during the second half of the liver stage developmental process. This process, which follows hepatocyte invasion by sporozoites, consists in karyokinesis without cytokinesis before budding off of thousands of uninucleate merozoites and lasts 50–70 hr in rodents.

Phenotype
The Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence MSP-1 expression specifically in liver stages. Removal of the FRTed 3'UTR of msp-1  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 FRTed 3'UTR of msp-1. Removal of the 3'UTR region of msp-1 resulted in 'down-regulation' of expression of MSP-1. Quantification of MSP-1 expression in mature liver stages showed that mutant parasites produced 6.5% of the amounts of MSP-1 synthesized by control parasites (see 'Additional information'). This indicated that MSP-1 was silenced in the 'excised' MSP1/Cond mutant liver stages. Because the proportion of non-excised parasites (5%) was similar to the proportion of residual MSP-1 (6.5%) in the parasite population, the excised parasites appear to produce virtually no MSP-1.
The liver stages of the mutant yielded on average 30-fold fewer merozoites than wild type liver stages and mature liver stages showed aberrant merozoite formation (see also 'Additional remarks'), indicating that MSP-1 plays a crucial role in the formation of merozoites by the liver stage of the parasite.

Additional information
In the paper unsuccessful attempts  to inactivate MSP-1 in erythrocytic stages by disruption of msp-1 using standard transfection technology  are mentioned (see RMgm-326), indicating the essential nature of MSP-1 for P. berghei blood stage multiplication.

To verify that the excised MSP1/Cond liver stages did not produce MSP1, fluorescent HepG2 cells containing MSP1/Cond or control parasites were sorted by FACS after 58 hr incubation, and the cell extracts were analyzed by western blotting using anti-MSP-1 antibodies. Quantification of the MSP-1 signal showed that mutant parasites produced 6.5% of the amounts of MSP1 synthesized by control parasites. This indicated that MSP-1 was silenced in excised MSP1/Cond mutant liver stages. Because the proportion of non-excised parasites (5%) was similar to the proportion of residual MSP-1 (6.5%) in the parasite population, the excised parasites appear to produce virtually no MSP1.

Liver stage development was investigated by using transmission electron microscopy (TEM). HepG2 cells were incubated with mutant and control sporozoites, and after 58 hr incubation, fluorescent cells were sorted by FACS and processed for TEM. The multinucleated control parasites generated merozoites of homogenous size and regular shape that budded off from the periphery of the parasite mass, progressively filling the parasitophorous vacuole and ultimately the host cell cytoplasm. By contrast, the majority of MSP1/Cond parasites contained numerous vacuoles of various sizes and generated abnormal progeny. Merozoite buds emerged within the intraparasitic vacuoles, rather than at the parasite periphery, and the internal buds displayed abnormal shapes, frequently having an enlarged base or an orientation tangential rather than perpendicular to the plane of the parasite membrane. these results indicate that MSP-1 appears to play a crucial role in the formation of merozoites by the liver stage of the parasite.

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.

Other mutants
RMgm-201: A mutant expressing a mutated form of MSP-1 (Replacement of the P. berghei MSP-1 19 kD C-terminal with the P. falciparum MSP-1 19 kD C-terminal)
RMgm-326: Unsuccessful attempts to disrupt MSP-1


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0831000
Gene Model P. falciparum ortholog PF3D7_0930300
Gene productmerozoite surface protein 1
Gene product: Alternative nameMSP-1, MSP1
Details of the genetic modification
Short description of the mutationThe mutant contains a FRTed 3'UTR of the msp-1 locus
Inducable system usedFlp/FRT
Short description of the conditional mutagenesisThe 3'UTR of msp1 is removed in the sporozoite stage by the Flp/FRT system.
Additional remarks inducable system
Click to view information
Click to hide information
This mutant has been generated by replacement of the endogenous msp-1 gene by an msp-1 gene with a FRTed 3' UTR in mutant RMgm-269 that expresses Flp and GFP.
Removal of the FRTed 3'UTR of msp-1 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 3'UTR of msp-1 which was flanked by FRT sequences.
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe targeting plasmids used in this study contain the human dihydrofolate reductase (hDHFR) marker, which is expressed via the EF1α promoter and the 3′ regulatory sequences of the DHFR-TS gene of P. berghei, the pUC18 backbone, and targeting DNA from P. berghei NK65. Plasmid pMSP1/FRT contains (1) the last 0.6 kb of the MSP1 coding sequence immediately followed by the first 16 nucleotides of the TRAP 3′ regulatory region, (2) a first FRT site, (3) the TRAP 3′ regulatory region (0.6 kb) starting at position +16 after the TRAP stop codon, (4) the hDHFR cassette, (5) the second FRT site, (6) the plasmid backbone, and (7) 0.6 kb of MSP1 3′ regulatory sequence.
Additional remarks selection procedureThe construct for introducing the FRTed 3'UTR of the msp1 gene has been transfected into mutant RMgm-269 that already contains the pyrimethamine resistance pbdhfr selectable marker (used for introducing the gfp gene). Therefore, to introduce this construct the hdhfr selectable marker is used in conjunction with WR92100 selection.
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 15'-CCCAAGCTTATAAATTATTGAAATATTTGTTGG-3'
Additional information primer 1P23 (3' part (580 bp) of the MSP1 coding sequence)
Sequence Primer 25'-cgcgcatgccaaaacatatacaactttttttaaaccc-3'
Additional information primer 2P24 (3' part (580 bp) of the MSP1 coding sequence)
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 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 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 the 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 15'- GCGCCCATGGTTTATTCAGACGTAATAATTATGTGCTGAAAGG-3'
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_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