RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1505

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1349800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein \| sporozoite surface protein 2 (sporozoite surface protein 2; SSP2; SSP-2; TRAP) Name tag: GFP
Phenotype	Sporozoite;

Last modified: 20 July 2016, 11:16

*RMgm-1505

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) : 27427910
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Kehrer J; Mair GR
Name Group/Department	Integrative Parasitology, Center for Infectious Diseases
Name Institute	University of Heidelberg Medical School
City	Heidelberg
Country	Germany
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Name of the mutant parasite
RMgm number	RMgm-1505
Principal name	ss::gfp::trap
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	A faint, speckled surface signal in non-permeabilized sporozoites. SS::GFP::TRAP is expressed in midgut and salivary gland sporozoites with a localisation consistent with wildtype TRAP. Sporozoites are motile.
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses an N-terminal GFP-tagged version of TRAP Protein (function) TRAP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). The cytoplasmic part (tail) of the protein is postulated to interact with actin–myosin motor proteins giving the force needed for motility. TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells as has been shown by analysis of mutant parasites lacking expression of TRAP. Analyses of mutants that express TRAP with mutated forms of the cytoplasmic tail have shown the role of this domain for gliding motility and invasion of host cells. Phenotype A faint, speckled surface signal in non-permeabilized sporozoites. SS::GFP::TRAP is expressed in midgut and salivary gland sporozoites with a localisation consistent with wildtype TRAP. Sporozoites are motile. Additional information Other mutants See the link PF3D7_1335900 for other TRAP mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1349800

Gene Model P. falciparum ortholog

PF3D7_1335900

Gene product

thrombospondin-related anonymous protein | sporozoite surface protein 2

Gene product: Alternative name

sporozoite surface protein 2; SSP2; SSP-2; TRAP

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

N-terminal

Additional remarks: tagging

To tag TRAP with GFP at the N-terminal after signal peptide cleavage, 1054 bp of the 5’UTR of trap together with the first 105 bp of the coding region were cloned in front of GFP linked by a 6-amino acid linker. This was followed by an 8-amino acid linker, the rest of the coding sequence of trap, followed by 514 bp of the 3’ UTR region of dhfs, followed by a hdhfr selection cassette. The pasmid was linearized with BstBI within the TRAP 5’UTR resulting in introduction of SS::GFP::TRAP by single crossover integration.

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) 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

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

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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