RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5237

Malaria parasite	P. berghei
Genotype
Disrupted	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)
Phenotype	Sporozoite;

Last modified: 12 June 2023, 13:59

*RMgm-5237

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) : 37306042
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	Braumann F, Frischknecht F
Name Group/Department	Integrative Parasitology, Center for Infectious Diseases
Name Institute	Heidelberg University Medical School
City	Heidelberg
Country	Germany
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Name of the mutant parasite
RMgm number	RMgm-5237
Principal name	trap(-)
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 analysed in detail: - Normal oocyst production with sporozoite formation. Sporozoites are released in the hemocoel but are impaired in salivary gland invasion. No (strongly reduced) salivary gland sporozoite numbers. - The mutant lacks expression of TRAP. The trap gene has been deleted and does not contain a drug-selectable marker (that has been removed using negative selection with 5-FC). This mutant is used to introduce mutated forms of the trap gene.
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of TRAP. The trap gene has been deleted and does not contain a drug-selectable marker (that has been removed using negative selection with 5-FC). This mutant is used to introduce mutated forms of the trap gene. Published in bioRxiv preprint doi: https://doi.org/10.1101/2022.08.24.505106 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). TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells. Phenotype Not analysed in detail: - Normal oocyst production with sporozoite formation. Sporozoites are released in the hemocoel but are impaired in salivary gland invasion. No (strongly reduced) salivary gland sporozoite numbers. - The mutant lacks expression of TRAP. The trap gene has been deleted and does not contain a drug-selectable marker (that has been removed using negative selection with 5-FC). This mutant is used to introduce mutated forms of the trap gene. Additional information Other mutants

Disrupted: Mutant parasite with a disrupted 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

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Yes

Name of PlasmoGEM construct/vector

PbGEM-107890

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

Selectable marker used to select the mutant parasite

hdhfr/yfcu

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

5-fluorocytosine (5-FC)

Additional remarks genetic modification

TRAP knockout (trap(-)) parasites were generated with the PlasmoGem vector PbGEM-107890 using standard protocols. Isogenic trap(-) parasites were subsequently negatively selected with 5-fluorocytosine (1.0 mg/mL in the drinking water) to give rise to selection marker-free trap(-) parasites.

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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