RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-25

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_0622921; Gene model (P.falciparum): Not available; Gene product: 18S ribosomal RNA (small subunit (ssu) of the d-type ribosomal rna gene unit)
Phenotype	Oocyst; Sporozoite;

Last modified: 28 August 2015, 16:36

*RMgm-25

Successful modification	The 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	MRA-441
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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The mutant parasite was generated by
Name PI/Researcher	R.M.L. van Spaendonk, C.J. Janse, A.P. Waters
Name Group/Department	Leiden Malaria Research Group
Name Institute	Leiden University Medical Center
City	Leiden
Country	The Netherlands
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Name of the mutant parasite
RMgm number	RMgm-25
Principal name	133cl2
Alternative name	Drep2
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	Not different from wild type
Oocyst	Small growth delay (retardation) of oocysts
Sporozoite	Possibly less sporozoites are produced as a result of the growth delay of the oocysts. Sporozoites are infectious to mice.
Liver stage	Not 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 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. Other mutants Two other mutants has been generated that lack expression of d-type ribosomal RNA. Mutant RMgm-24 in which 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. Mutant RMgm-23 contains a disruption of the ssu (small subunit) part of the rRNA locus (disruption by single cross-over integration). 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

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Details of the target gene

Gene Model of Rodent Parasite PBANKA_0622921

Gene Model P. falciparum ortholog Not available

Gene product 18S ribosomal RNA

Gene product: Alternative name small subunit (ssu) of the d-type ribosomal rna gene unit

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Details of the genetic modification

Inducable system used No

Additional remarks inducable system

Type of plasmid/construct used (Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used No

Modified PlasmoGEM construct/vector used No

Plasmid/construct map

Plasmid/construct sequence

not available

Restriction sites to linearize plasmid

Partial or complete disruption of the gene Partial

Additional remarks partial/complete disruption Disruption 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).

Selectable marker used to select the mutant parasite pbdhfr

Promoter of the selectable marker pbdhfr

Selection (positive) procedure pyrimethamine

Selection (negative) procedure No

Additional remarks genetic modification Disruption 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).

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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