RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5234
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0209900; Gene model (P.falciparum): PF3D7_0103200; Gene product: nucleoside transporter 4 (NT4)
Transgene
Transgene not Plasmodium: mTurquoise2 fluorescent protein
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0209900; Gene product: nucleoside transporter 4 (NT4)
Replacement locus: Gene model: PBANKA_0209900; Gene product: nucleoside transporter 4 (NT4)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 2 September 2022, 14:10
  *RMgm-5234
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 36003062
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherDeveci G, Aly ASI
Name Group/DepartmentAly Lab, Beykoz Institute of Life Sciences and Biotechnology
Name InstituteBezmialem Vakif University
CityIstanbul
CountryTurkey
Name of the mutant parasite
RMgm numberRMgm-5234
Principal namePbnt4(-)
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
OocystWildtype-like oocyst production. Evidence is presented for a reduced (delayed) egress of sporozoites from the oocysts and a defect in salivary gland invasion. Strongly reduced numbers of salivary gland sporozoites.
SporozoiteEvidence is presented for a reduced (delayed) egress of sporozoites from the oocysts and a defect in salivary gland invasion. Strongly reduced numbers of salivary gland sporozoites.
Mice injected with wild-type sporozoites showed a prepatent period of 4 days and 7 days for salivary glands sporozoites and hemolymph sporozoites, respectively, and they did not survive due to high parasitemia beyond days 16 and 18 post sporozoite injection, respectively. However, all mice injected with knockout sporozoites did not develop any blood-stage parasitemia till day 21 post sporozoite injection.
Liver stageMice injected with wild-type sporozoites showed a prepatent period of 4 days and 7 days for salivary glands sporozoites and hemolymph sporozoites, respectively, and they did not survive due to high parasitemia beyond days 16 and 18 post sporozoite injection, respectively. However, all mice injected with knockout sporozoites did not develop any blood-stage parasitemia till day 21 post sporozoite injection.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of NT4 and expresses the reporter mTurquoise2 fluorescent protein under control of the eef1a promoter

Protein (function)
Plasmodium is a purine-auxotrophic parasite that lacks de novo purine synthesis and depends on the host to get the required purines. Parasites contain a special class of protein family called as equilibirative nucleoside transporter (ENT) which are central in the transport of nucleoside from host to parasite. Four members (NT1, NT2, NT3, and NT4) of the NT gene family have been identified in the genome of Plasmodium species, which are known to play important roles in purines acquisition from the host.

Phenotype
Wildtype-like oocyst production. Evidence is presented for a reduced (delayed) egress of sporozoites from the oocysts and a defect in salivary gland invasion. Strongly reduced numbers of salivary gland sporozoites.Mice injected with wild-type sporozoites showed a prepatent period of 4 days and 7 days for salivary glands sporozoites and hemolymph sporozoites, respectively, and they did not survive due to high parasitemia beyond days 16 and 18 post sporozoite injection, respectively. However, all mice injected with knockout sporozoites did not develop any blood-stage parasitemia till day 21 post sporozoite injection.


Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0209900
Gene Model P. falciparum ortholog PF3D7_0103200
Gene productnucleoside transporter 4
Gene product: Alternative nameNT4
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
Selectable marker used to select the mutant parasitehdhfr
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemTurquoise2 fluorescent protein
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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_0209900
Gene productnucleoside transporter 4
Gene product: Alternative nameNT4
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_0209900
Gene productnucleoside transporter 4
Gene product: Alternative nameNT4
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