RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-56

Malaria parasite	P. berghei
Genotype
Mutated	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) Details mutation: Mutation in the cytoplasmic tail (lacking the 37 carboxy-terminal residues)
Phenotype	Sporozoite; Liver stage;

Last modified: 4 March 2010, 23:39

*RMgm-56

Successful modification	The parasite was generated by the genetic modification
The mutant contains the following genetic modification(s)	Gene mutation
Reference (PubMed-PMID number)	Reference 1 (PMID number) : 10579715
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	S. Kappe, V. Nussenzweig, R. Menard
Name Group/Department	Department of Pathology, Kaplan Cancer Center
Name Institute	New York University School of Medicine
City	New York
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-56
Principal name	T∆L
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	Normal numbers of midgut sporozoites are formed. Evidence is presented that the cell surface localization of TRAP is not affected. Typical gliding motility is absent. No infection of HepG2 cells in vitro. No infection of rats after intravenous inoculation of sporozoites.
Liver stage	No infection of HepG2 cells in vitro. No infection of rats after intravenous inoculation of sporozoites.
Additional remarks phenotype	Mutant/mutation The mutant expresses a mutated form of TRAP (thrombospondin-related anonymous protein). The mutated gene lacks the 37 carboxy-terminal residues of the cytoplasmic tail. 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 (RMgm-47; RMgm-53). Phenotype Analysis of the phenotype of the mutant with a mutated cytoplasmic tail (see 'Additional remarks genetic modification') indicates that the mutation does not affect cell surface localization of TRAP, indicating that the cytoplasmic tail is dispensable for surface exposure of the protein. The mutation affects motility, cell invasion and infectivity of sprozoites. The role of the different domains of the protein in motility and invasion has been analysed in other mutant parasites expressing mutated forms of TRAP (see below). Other mutants P. berghei mutants have been generated with mutated cytoplasmic tails of TRAP (RMgm-54; RMgm-55; RMgm-57; RMgm-149, RMgm-150, RMgm-151). Other P. berghei mutants have been generated with mutated extracellular (adhesive) domains (A-domain: RMgm-48; RMgm-49; TSR domain: RMgm-50; RMgm-51; A-domain and TSR domain: RMgm-52). A P. berghei mutant has been generated in which the endogenous P. berghei trap was replaced with (mutated forms of) P. falciparum trap (RMgm-58)

Mutated: Mutant parasite with a mutated 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

Short description of the mutation

Mutation in the cytoplasmic tail (lacking the 37 carboxy-terminal residues)

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

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

pbdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The construct used results in 'disruption of the wild type trap-gene and introduction of a full length mutated trap gene under control of the wild type regulatory (3'UTR, 5'UTR) sequences.
The mutated gene lacks the 37 carboxy-terminal residues of the cytoplasmic tail.

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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