RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-161

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0911700; Gene model (P.falciparum): PF3D7_1136900; Gene product: subtilisin-like protease 2 (SUB2) Name tag: c-myc
Phenotype	Asexual bloodstage;

Last modified: 24 February 2009, 15:58

*RMgm-161

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene tagging
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 14678331
MR4 number
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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
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The mutant parasite was generated by
Name PI/Researcher	P. Uzureau; C.J. Janse; A.P. Waters; C. Braun Breton
Name Group/Department	Unité de Biologie des Interactions Hôte-Parasite
Name Institute	Institut Pasteur
City	Paris
Country	France
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Name of the mutant parasite
RMgm number	RMgm-161
Principal name	sub2
Alternative name	wt-TmDX
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Normal growth and multiplication of the asexual blood stages. IFA analysis using cmyc antibodies shows expression of SUB2 in schizonts/merozoites.
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a c-myc tagged form of SUB2. Protein (function) SUB2 is a subtilisin-like serine protease. In Plasmodium three subtilisin-like proteases (SUB1-3) have been identified. SUB2 has been shown to be associated with dense granules and/or micronemes, the contents of which are released during erythrocyte invasion. It has been proposed to function as a 'sheddase', involved in the release of MSP1 and AMA1 during invasion (Harris et al., 2005, PloS Pathogens, 3, e29) Attempts to disrupt the sub2 gene (in P. berghei and P. falciparum) were not successful, indicating an essential function in the blood stages (see RMgm-160). Phenotype The mutant shows normal growth and multiplication of the asexual blood stages, indicating that the cmyc-tagging does not affect the function of SUB2. IFA analysis using cmyc antibodies shows expression of SUB2 in schizonts/merozoites. Additional information Attempts to disrupt the sub2 gene were not successful, indicating an essential function in the blood stages (see RMgm-160). See also GenBank accession numbers AJ242629 and AF145052 for the correct sequence of the gene encoding SUB2 Other mutants RMgm-160: Unsuccessful attempts to disrupt sub2

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0911700

Gene Model P. falciparum ortholog

PF3D7_1136900

Gene product

subtilisin-like protease 2

Gene product: Alternative name

SUB2

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Details of the genetic modification

Name of the tag

c-myc

Details of tagging

C-terminal

Additional remarks: tagging

Multiple epitope tag, called TrimycDuoXpress tag (TmDX tag). The TmDX tag consists of 56 amino acids corresponding to three c-myc epitopes separated by two Xpress epitopes . Each c-myc and Xpress epitope is separated by a hinge composed of two alanine or two valine residues

Commercial source of tag-antibodies

Type of plasmid/construct

Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

BsmI

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

A multiple epitope tag, called TrimycDuoXpress tag (TmDX tag) was used. The TmDX tag consists of 56 amino acids corresponding to three c-myc epitopes separated by two Xpress epitopes. Each c-myc and Xpress epitope is separated by a hinge composed of two alanine or two valine residues. The BsmI linearized pSub2-wt-TmDX construct was designed to integrate via a double crossing-over event, resulting in the insertion of the TmDX tag in frame with the C-terminus of the SUB2 protein and the insertion of the Toxoplasma gondii DHFR-TS selectable marker.
For more detailed information about plasmid construction and primer sequences, see 'Experimental Procedures' (Uzureau et.al., Cellular Microbiology (2003) 6:65-78)

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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