RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-217
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0819900; Gene model (P.falciparum): PF3D7_0919000; Gene product: nucleosome assembly protein (NAPS, nucleosome assembly protein S; SET)
Name tag: GFP
Transgene
 Transgene Plasmodium: Gene model: PBANKA_0819900; Gene model (P.falciparum): PF3D7_0919000; Gene product: nucleosome assembly protein (NAPS, nucleosome assembly protein S; SET)
Promoter: Gene model: PBANKA_0819900; Gene model (P.falciparum): PF3D7_0919000; Gene product: nucleosome assembly protein (NAPS, nucleosome assembly protein S; SET)
3'UTR: Gene model: PBANKA_0819900; Gene product: nucleosome assembly protein (NAPS, nucleosome assembly protein S; SET)
Insertion locus: Gene model: Not available; Gene product: Not available (small subunit ribosomal rna gene (c-or d-type unit))
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 2 August 2011, 10:07
  *RMgm-217
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 16677299
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 8417HP
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (Janse et al., Exp. Parasitol. 68, 274-282).
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The mutant parasite was generated by
Name PI/ResearcherT. Pace, M. Ponzi
Name Group/DepartmentDipartimento di Malattie Infettive Parassitarie ed Immunomediate
Name InstituteIstituto Superiore di Sanità
CityRome
CountryItaly
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Name of the mutant parasite
RMgm numberRMgm-217
Principal nameG-SET/GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNo expression is observed in the asexual blood stages, except for a very small fraction of (mature) schizonts
Gametocyte/GameteGametocytes show (strong) GFP expression with a significant higher expression levels (20 fold) in male gametocytes compared to female gametocytes. GFP fluorescence is localized in the nucleus
Fertilization and ookineteLow NAPS/SET-GFP expression in the nucleus of ookinetes
OocystNo NAPS/SET-GFP expression in oocysts
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a c-terminal GFP-tagged form of NAPS/SET. The gfp-naps/set gene is under the control of the 'gametocyte-specific promoter' (G-promoter) of naps/set (see 'Phenotype'). The gfp-naps/set gene is integrated into the non-essential c- or d-type ribosomal rna locus and has its own 3'-UTR region.
See also RMgm-218 for a mutant expressing gfp-naps/set gene under the control of the 'asexual blood stage promoter' (A-promoter) of naps/set (see 'Phenotype').

Protein (function)
Plasmodium contains two nucleosome assembly proteins, termed NAPL and NAPS (NAPS: PF1093c nucleosome binding protein; PBANKA_081990; NAPL: PFL0185c Nucleosome binding protein 1; PBANKA_060270). NAPL is predominantly localized in the cytoplasm, whereas NAPS has mainly a nuclear location. Both NAPS and NAPL interact with histones but only NAPS is able to deposit histones onto DNA.

Phenotype
The single-copy gene encoding NAPS/SET  produces mRNA transcripts in asexual blood stage parasites with a different size of the 5'UTR region compared with transcripts produced in gametocytes (Pace et al., 1998, Mol Biochem Parasitol. 97:45-53). This size difference is the result of post-transcriptional processing and the use of alternative transcription initiation sites. The phenotype analyses of this mutant and mutant RMgm-218  demonstrates the presence of two distinct promoters upstream of the naps/set gene, the one active in all asexual blood stage parasites (A-promoter) while the other is active only in gametocytes(G-promoter). In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the A-promoter.

Additional information
GenBank Accession No. of naps/set: AF031904.
Attempts to disrupt NAPL and NAPS of P. falciparum (Gill et al., 2009, J Biol Chem. 2009, Jan 27; Gill J. et al, 2010 Malar J) and NAPS of P. berghei (RMgm-246) were not successful, indicating an essential role during asexual blood stage development.

Other mutants
RMgm-218: A mutant expressing a c-terminal GFP-tagged form of NAPS/SET under the control of the 'asexual blood stage promoter' (A-promoter) and introduced into the c/d-rrna locus
RMgm-246: Unsuccessful attempts to disrupt NAPS/SET

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0819900
Gene Model P. falciparum ortholog PF3D7_0919000
Gene productnucleosome assembly protein
Gene product: Alternative nameNAPS, nucleosome assembly protein S; SET
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Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 ApaI
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe single-copy gene encoding NAPS/SET produces mRNA transcripts in asexual blood stage parasites with a different size of the 5'UTR region compared with transcripts produced in gametocytes (Pace et al., 1998, Mol Biochem Parasitol. 97:45-53). This size difference is the result of post-transcriptional processing and the use of alternative transcription initiation sites. Two different transfection constructs have been generated that contained a C-terminal fusion between the entire coding region of the naps/set gene and the gfp reporter. Set/gfp was placed under the control of two of distinct promoter regions, i.e. the 1.3 kb fragment upstream of the gametocyte-specific mRNA (G-promoter; the distal promoter) or a 1.3 kb fragment upstream of the asexual mRNA (promoter-A; the proximal promoter) and the same 3'UTR. The two constructs (pG-SET/GFP and pA-SET/GFP respectively) also contained the P. berghei dhfr-ts as a selectable marker cassette and a fragment of the small subunit of the d-rrna gene of P. berghei for targeted integration into the non-essential c or d-rrna genes by a single-cross over event.
Construct pG-SET/GFP was used to generate the mutant described here and construct pA-SET/GFP was used to generate mutant RMgm-218
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 1GAATTCATGAAGAGAGATCGTTCAGA
Additional information primer 1Set coding region (EcoRI)
Sequence Primer 2CCCGGGATCATCATTATCTTTTCCATC
Additional information primer 2Set coding region (XmaI)
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PBANKA_0819900
Gene Model P. falciparum ortholog PF3D7_0919000
Gene productnucleosome assembly protein
Gene product: Alternative nameNAPS, nucleosome assembly protein S; SET
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
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 ApaI
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe single-copy gene encoding NAPS/SET produces mRNA transcripts in asexual blood stage parasites with a different size of the 5'UTR region compared with transcripts produced in gametocytes (Pace et al., 1998, Mol Biochem Parasitol. 97:45-53). This size difference is the result of post-transcriptional processing and the use of alternative transcription initiation sites. Two different transfection constructs have been generated that contained a C-terminal fusion between the entire coding region of the naps/set gene and the gfp reporter. Set/gfp was placed under the control of two of distinct promoter regions, i.e. the 1.3 kb fragment upstream of the gametocyte-specific mRNA (G-promoter; the distal promoter) or a 1.3 kb fragment upstream of the asexual mRNA (promoter-A; the proximal promoter) and the same 3'UTR. The two constructs (pG-SET/GFP and pA-SET/GFP respectively) also contained the P. berghei dhfr-ts as a selectable marker cassette and a fragment of the small subunit of the d-rrna gene of P. berghei for targeted integration into the non-essential c or d-rrna genes by a single-cross over event.
Construct pG-SET/GFP was used to generate the mutant described here and construct pA-SET/GFP was used to generate mutant RMgm-218
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0819900
Gene Model P. falciparum ortholog PF3D7_0919000
Gene productnucleosome assembly protein
Gene product: Alternative nameNAPS, nucleosome assembly protein S; SET
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 1GTCGACGATCCACACATATTTGTATGC
Additional information primer 1G-fragment (SalI)
Sequence Primer 2GAATTCTGGGGATATTACATATAGTCTT
Additional information primer 2G-fragment (EcoRI)
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3'-UTR
Gene Model of Parasite PBANKA_0819900
Gene productnucleosome assembly protein
Gene product: Alternative nameNAPS, nucleosome assembly protein S; SET
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 1ACTAGTGATGATTAATAAATAAATTGATC
Additional information primer 13'UTR (SpeI)
Sequence Primer 2GCGGCCGCGATATCCCTCTAAGTAGCATT
Additional information primer 23'UTR (NotI)
Insertion/Replacement locus
Replacement / InsertionInsertion locus
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c-or d-type unit)
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 1GCGGCCGCCGGTCCAACCTGGTTGATCTTGCCAG
Additional information primer 1SSU (NotI)
Sequence Primer 2GAGCTCACTACGTGACCTACGGAAACCTTGTTACG
Additional information primer 2SSU (SacI)
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren