RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5051

Malaria parasite	P. yoelii
Genotype
Disrupted	Gene model (rodent): PY17X_0415800; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP)
Phenotype	Fertilization and ookinete; Oocyst;

Last modified: 29 July 2021, 14:17

*RMgm-5051

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) : 34301597
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Yang Z, Yuan J
Name Group/Department	State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name Institute	Xiamen University
City	Xiamen, Fujian
Country	China
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Name of the mutant parasite
RMgm number	RMgm-5051
Principal name	Δctrp
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	Not tested
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Ookinetes are formed but no traversal through mosquito midgut cells. Motility of ookinetes is impaired.
Oocyst	Ookinetes are formed but no traversal through mosquito midgut cells. Motility of ookinetes is impaired.
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of CTRP The mutant has been generated using CRISPR/cas9 genome editing (using constructs as described for mutant RMgm-1095 Protein (function) CTRP 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). CTRP has six tandemly arrayed A domains followed by seven TSP type 1-like domains. CTRP is located in the micronemes of ookinetes. This structure is similar to that of TRAP (PF13_0201; PB000374.03.0), a malaria sporozoite protein critical for sporozoite motility and invasion into host cells. CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells. Phenotype Ookinetes are formed but no traversal through mosquito midgut cells. Motility of ookinetes is impaired. 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

PY17X_0415800

Gene Model P. falciparum ortholog

PF3D7_0315200

Gene product

circumsporozoite- and TRAP-related protein

Gene product: Alternative name

CTRP

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

CRISPR/Cas9 construct: integration through double strand break repair

PlasmoGEM (Sanger) construct/vector used

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

Additional remarks genetic modification

CRISPR-Cas9 plasmid pYCm was used for all the parasite genetic modification. To construct vectors for gene editing, we amplified 5′ and 3′ genomic sequence (400 to 500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. Oligonucleotides for single guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. DNA fragments encoding 6HA, 4Myc, 3V5, and GFP or mCherry were inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. Infected red blood cells (iRBCs) were electroporated with 5 to 10 μg of plasmid DNA using the Lonza Nucleofector . Transfected parasites were immediately intravenously injected into a naïve mouse and were exposed to pyrimethamine (6 mg/ml) 24 hours after transfection.

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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