Summary

RMgm-24
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0622961; Gene model (P.falciparum): Not available; Gene product: 28S ribosomal RNA (large subunit (lsu) of the d-type ribosomal rna gene unit)
Phenotype Oocyst; Sporozoite;
Last modified: 28 August 2015, 16:37
  *RMgm-24
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 11292830
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherR.M.L. van Spaendonk, C.J. Janse, A.P. Waters
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-24
Principal name117cl4
Alternative nameDrep1
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
OocystSmall growth delay (retardation) of oocysts
SporozoitePossibly less sporozoites are produced as a result of the growth delay of the oocysts.
Sporozoites are infectious to mice.
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of  d-type ribosomal rna.

Gene (function)
P. berghei contains four distinct copies of the ribosomal RNA gene units (A-D), each encoding the following rRNA molecules: large subunit (LSU; 28 S) rRNA, small subunit (SSU; 18 S) rRNA and 5.8 S rRNA .
Based on differences in sequence and expression the four gene units are divided into the blood stage A-type (a- and b-unit) and S-type (c- and d-units), which is transcribed mainly in the proliferative stages in the mosquito.  The A-type ribosomes are present in the liver and blood stages of the parasite, and the S-type ribosomes are the predominant types produced during development in the mosquito.

Phenotype
The phenotype analyzes show that the lack of expression of d-type ribosomal RNA does have a relatively minor effect on parasite development, resulting in a delayed maturation of oocysts. The sporozoites formed are infectious to the mammalian host (Swiss mice). See also mutants RMgm-21 and RMgm-22 which lack expression of c-type ribosomal RNA.

Additional information
The d-type rRNA gene has been disrupted  by double cross-over recombination, resulting in deletion of the 5.8S, ITS2 region and part of the 5'-LSU.

Other mutants
Two other mutants has been generated that lack expression of d-type ribosomal RNA. Mutant RMgm-23 contains a disruption of the ssu (small subunit) part of the rRNA locus (disruption by single cross-over integration). In mutant RMgm-25 the d-type rRNA gene unit has been disrupted by double cross-over recombination, resulting in deletion of the SSU, ITS1, 5.8S, ITS2 region, and part of the D-ETS and 5'-LSU.These mutants shows a similar phenotype as described above.
Mutants have been generated lacking expression of the c-type ribosomal RNA. These mutants did not show a distinct phenotype (RMgm-21, RMgm-22).
Mutants have been generated that lack expression of both c- and d-type ribosomal RNA (pers. comm. B. Franke-Fayard; C.J. Janse). These mutants show a strong delayed maturation of the oocysts with reduced numbers of salivary gland sporozoites. Salivary gland sporozoites produced were infective to the mammalian host (Swiss mice).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0622961
Gene Model P. falciparum ortholog Not available
Gene product28S ribosomal RNA
Gene product: Alternative namelarge subunit (lsu) of the d-type ribosomal rna gene unit
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
Click to view information
Click to hide information
not available
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption Disruption by double cross-over recombination, resulting in deletion of the 5.8S, ITS2 region and part of the 5'-LSU.
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationDisruption by double cross-over recombination, resulting in deletion of the 5.8S, ITS2 region and part of the 5'-LSU.
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