RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4933
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0800500; Gene model (P.falciparum): PF3D7_1252200; Gene product: chitinase (CHT1)
Transgene
 Transgene not Plasmodium: mCherry
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)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 25 January 2021, 14:09
  *RMgm-4933
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 33489941
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 RMgm-1320
Other information parent lineThis transgenic reporter line (1868cl1; RMgm-1320) expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Both reporter cassetes are introduced into the silent 230p locus, using a single DNA construct. This transgenic line does not contain a drug-selectable marker
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The mutant parasite was generated by
Name PI/ResearcherPatra KP, Kaur H, Kolli SK, Janse CJ, Vinetz JM
Name Group/DepartmentSection of Infectious Diseases, Department of Internal Medicine
Name InstituteYale School of Medicine
CityNew 7 Haven, Connecticut
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-4933
Principal name3152cl1
Alternative namePbΔcht1; PbANKA-CHT1 GIMO
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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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
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of CHT1. In the mutant the endogenous P. berghei cht1 gene has been deleted by replacing the cht1 gene with the drug-selectable marker cassette hdhfr-yfcu. It also expresses mCherry and luciferase under constitutive promoters

Protein (function)
CHT1, a secreted protein, is expressed in the ookinete stage and located in the micronemes. It contains putative proenzyme and chitin-binding domains. Chitinases are enzymes that hydrolize chitin, a (1-4)-β homopolymer of N-acetylglucosamine.

The Plasmodium ookinete secreted chitinase(s) are endochitinases of family 18 glycohydrolases that enable the parasite to dissolve the chitin (N-acetylglucosamine polymer)-containing peritrophic matrix (PM) within the mosquito midgut, en route crossing the midgut epithelium. Plasmodium ookinete-secreted chitinases have been classified as short (PfCHT1, PgCHT2 and PrCHT1) and long form (PgCHT1, PvCHT1 and PbCHT1) based on the presence or absence of predicted proenzyme and chitin binding domains (CBD) at the N- and C-terminal of the protein, respectively where short forms do not possess either domain.

Phenotype
The phenotype has not been analysed in detail. This mutant has been used to introduce the cht1 gene of P. falciparum into the P. berghei genome under control of the Pbcht1 regulatory sequences (see RMgm-4934). PbΔcht1 produced ookinete and oocyst numbers that were in the range of wild type parasites.

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0800500
Gene Model P. falciparum ortholog PF3D7_1252200
Gene productchitinase
Gene product: Alternative nameCHT1
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption We deleted the Pbcht1 CDS by replacing it with the positive-negative selectable marker (SM) to create a P. berghei cht1 deletion GIMO line (PbΔcht1). To do this, we generated DNA construct pL2321, which is based on the standard GIMO DNA construct pL0034. This construct contains the positive-negative human dihydrofolate reductase::yeast cytosine deaminase and uridyl phosphoribosyl transferase (hdhfr::yfcu) selection marker (SM) cassette. The 5’ and 3’ targeting regions of Pbcht1 were amplified from P. berghei ANKA genomic DNA. Fragments were digested (with ApaI/SacII and KpnI/NotI, respectively) and ligated into vector pL0034 to obtain pL2321. For transfection, pL2321 was linearized with ApaI/NotI and the linear construct was introduced in parasites of the P. berghei ANKA reference line 1868cl1 using standard methods of transfection
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 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
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: not Plasmodium
Transgene namemCherry
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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Other details transgene
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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
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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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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
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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: not Plasmodium
Transgene nameLuciferase
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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
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3'-UTR
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 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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren