RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-300

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_0804300; Gene model (P.falciparum): PF3D7_0706700; Gene product: DNA mismatch repair protein MSH2, putative (MSH2-2)
Phenotype	No phenotype has been described

Last modified: 24 January 2010, 15:02

*RMgm-300

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) : 17583362
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei NK65
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	L. Bethke, D. Wirth
Name Group/Department	Department of Immunology and Infectious Diseases
Name Institute	Harvard School of Public Health
City	Boston
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-300
Principal name	PbMSH2-2 mutant (clone A2, B2)
Alternative name
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	Not different from wild type
Sporozoite	Not different from wild type
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation In the mutant the gene encoding the mismatch repair protein MSH2-2 has been disrupted using an insertion plasmid designed to integrate via a single cross-over event. This integration event resulted in the presence of two truncated copies of the gene. Protein (function) MSH2-2 is a putative mismatch repair (MMR) protein. The most well characterized MMR pathway is the methyl-directed MutHLS system of Escherichia coli. Yeast, humans, and other eukaryotes have multiple homologs of bacterial MutS (MSH) and MutL (MLH), but no MutH. In the genome of P. falciparum two homologs of of MSH2 (bacterial MutS homolog) have been identified PfMSH2-1 (PF14_0254) and PfMSH2-2 (MAL7P1.206). Phenotype The phenotype analyses indicate a non-essential role of MSH2-2 during blood stage development, mosquito development and development in the liver under normal growth conditions (see also 'Additional information'). Additional information The mutant has been analysed in 'drug resistance' and 'microsatelaite instability' assays. These analyses did not provide evidence that disruption of MSH2-2 results in a strong mutator phenotype which may suggest that PbMSH2-2 is a minor MMR enzyme or that its function overlaps with that of MSH2-1 or other Plasmodium proteins. In the same study three other genes encoding putative mismatch repair (MMR) proteins have been targeted for disruption in P. berghei, i.e. PB300947.00.0 (PF14_0254; MSH2-1; DNA mismatch repair protein Msh2p, putative),, PB001297.02.0 (PFE0270c; MSH6; DNA repair protein, putative) and PB000037.02.0 (PF11_0184; MLH1; DNA mismatch repair protein MLH1, putative; mutL homolog). Generation and selection of mutants containing disrupted genes failed for all three genes, suggesting an essential role of these proteins during blood stage development. No information is provided on the (sequence of) DNA constructs used for disruption and on the number of attempts to disrupt these genes. Other mutants See RMgm-382, RMgm-383 and RMgm-384 for negative attempts to disrupt putative mismatch repair proteins

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0804300

Gene Model P. falciparum ortholog

PF3D7_0706700

Gene product

DNA mismatch repair protein MSH2, putative

Gene product: Alternative name

MSH2-2

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

Plasmid single cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

XcmI

Partial or complete disruption of the gene

Partial

Additional remarks partial/complete disruption

The plasmid designed to disrupt PbMSH2-2 included as a target sequence for homologous recombination, a 1380 bp fragment of PbMSH2-2, which lacked sequence encoding the first 127 amino acids of the protein, including a portion of the putative DNA binding domain, and was also missing the C-terminal 269 amino acids, including the ATP binding domain and the mutS signature motif.
The insertion plasmid was designed to integrate via a single crossover event, resulting in two truncated copies of the gene. No information is provided on the sequence of the primers used to amplify the target sequence for homologous recombination.

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

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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