RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4937
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1122300; Gene model (P.falciparum): PF3D7_0623400; Gene product: MEI2-like RNA-binding protein (Mei2)
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 (P230p)
Phenotype Liver stage;
Last modified: 4 December 2020, 17:13
  *RMgm-4937
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) Not published (yet)
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherOthman AS, Franke-Fayard B, Janse CJ
Name Group/DepartmentLeiden Malaria Group, Department of Parasitology
Name InstituteLeiden University Medical Center, LUMC
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-4937
Principal name2834cl2m1cl1
Alternative nameΔmei2
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
SporozoiteNot different from wild type
Liver stageNormal infectivity of sporozoites to hepatocytes in vitro and in vivo. Mutant parasites develop into large, mature liver stages with extensive nuclear division and expression of merozoite specific proteins, such as msp1 and ama1. Most of the parasites arrest growth, however, just before the formation of infective merozoites. Only after intravenously infection of mice with high doses of sporozoites (2x10(5)) some of the mice (3 out of 10) developed blood infections with a greatly prolonged prepatent period (12-14 days).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MEI2. It also expresses mCherry and luciferase under constitutive promoters. The mutant does not contain a drug-selectable marker.

Protein (function)
Mei2 is a member of the largest family of RNA binding proteins (RBPs) – those that contain a RNA recognition motif (RRM), a stretch of 70-90 amino acids that contain two consensus RNA-interacting motifs, RNP1 and RNP2. RRM-containing proteins are subdivided into ten separate families (RRM_1 thru RRM_10) based on shared amino acid identities between members of each family and Mei2 contains a C-terminal RRM_2, thought to be unique to fungi and plants.
Plasmodium contains a single Mei2-like gene

Phenotype
Normal blood stage and mosquito stage development.
Normal infectivity of sporozoites to hepatocytes in vitro and in vivo. Mutant parasites develop into large, mature liver stages with extensive nuclear division and expression of merozoite specific proteins, such as msp1 and ama1. Most of the parasites arrest growth, however, just before the formation of infective merozoites. Only after infection of mice with high doses of sporozoites (2x10(5)) some of the mice (3 out of 10) developed blood infections with a greatly prolonged prepatent period (12-14 days).

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1122300
Gene Model P. falciparum ortholog PF3D7_0623400
Gene productMEI2-like RNA-binding protein
Gene product: Alternative nameMei2
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 usedYes
Name of PlasmoGEM construct/vectorPbGEM-300555
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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor deletion of mei2, construct PbGEM-300555 (pL2206) was used (http://plasmogem.sanger.ac.uk/). This construct contains the hdhfr::yfcu selectable marker (SM) cassette with the P. berghei eef1α promoter region and 3’ terminal sequence of pbdhfr. Before transfection, the construct was linearized by digesting with NotI. Parasites of line 1868cl1 were transfected with construct pL2206 (exp. 2834) using standard transfection technologies and transformed parasites selected by positive selection with pyrimethamine. Selected parasites were cloned by limiting dilution and mutant 2834cl2 was used for genotype analysis and to generate the SM-free gene deletion mutant. To remove the hdhfr::yfcu SM cassette from the genomes of 2834cl2, these parasites were selected (negative selection) by treatment of infected mice with 5-Fluorocytosine (5-FC) as described. This treatment selects for parasites that have undergone homologous recombination between the two 3’-UTR of pbdhfr untranslated regions present in the integrated constructs pL2206, flanking the hdhfr::yfcu cassette and thereby removing the SM. Selection and cloning of the parasites resulted in the SM-free single gene-deletion mutant ∆mei2 (2834cl2m1cl1)
Additional remarks selection procedureSee above for applying negative selection to remove the drug selectable marker cassette
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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 modificationThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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 / 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
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
Other details transgene
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
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 nameP230p
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