RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1108

Malaria parasite	P. yoelii
Genotype
Tagged	Gene model (rodent): PY17X_1427200; Gene model (P.falciparum): PF3D7_0812300; Gene product: sporozoite surface protein 3, putative (SSP3) Name tag: 4xMyc and 3xHA
Phenotype	Sporozoite;

Last modified: 29 August 2014, 21:47

*RMgm-1108

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) : 25156733
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. yoelii
Parent strain/line	P. y. yoelii 17XNL
Name parent line/clone	cl1.1
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Harupa A; Kappe SHI
Name Group/Department	Seattle Biomedical Research Institute
Name Institute	Seattle Biomedical Research Institute
City	Seattle
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-1108
Principal name	Py SSP3nHa-cMyc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not tested
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Expression of the tagged version of SSP3 at the surface of sporozoites
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a tagged version of of sporozoite surface protein 3, putative (SSP3) (C-terminal quadruple Myc (4xMyc) tag and an internal triple HA (3xHA) tag). Protein (function) SSP3 is a predicted type 1 transmembrane protein. BLASTP identified orthologs in all Plasmodium species, but did not yield any potential orthologs in other apicomplexan or non-apicomplexan organisms. SSP3 contains thirteen highly conserved cysteine residues, twelve of which are within the predicted ectodomain and one within the predicted transmembrane domain. Structural homology searches suggested that the N-terminal region of SSP3 is similar to the s48/45 domain of Plasmodium 6-Cys proteins. Phenotype A mutant lacking expression of SSP3 (RMgm-1107) showed a defect in gliding motility of sporozoites. Analysis of the mutant expressing Myc/HA-tagged SSP3 provided evidence for a localization of SSP3 at the surface of sporozoites (no detection of the protein in blood stages). Additional information Evidence is presented for a surface localisation on sporozoites (but is not shed in gliding trails during gliding motility). Evidence is provided that SSP3 might be masked by CSP and is exposed and accessible to antibodies once CSP is shed. Other mutants A mutant lacking expression of SSP3 (RMgm-1107)

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_1427200

Gene Model P. falciparum ortholog

PF3D7_0812300

Gene product

sporozoite surface protein 3, putative

Gene product: Alternative name

SSP3

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Details of the genetic modification

Name of the tag

4xMyc and 3xHA

Details of tagging

C-terminal

Additional remarks: tagging

C-terminal quadruple Myc (4xMyc) tag and an internal triple HA (3xHA) tag

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) 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

Selectable marker used to select the mutant parasite

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

No details (sequence) are provided for the primers used.

Transgenic parasites were generated via double crossover homologous recombination to replace the endogenous open reading frame with the tagged version of SSP3. Expression of SSP3nHA-cMyc was controlled by its endogenous
promoter and the commonly used 3’ regulatory elements of the P. berghei DHFR/TS gene.

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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