RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-5366

Malaria parasite	P. yoelii
Genotype
Disrupted	Gene model (rodent): PY17X_1004800; Gene model (P.falciparum): PF3D7_0405700; Gene product: lysine decarboxylase, putative (amino acid decarboxylase, AAD; UIS14)
Phenotype	Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;

Last modified: 20 June 2023, 17:12

*RMgm-5366

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) : 37260388
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	Kina UY, Aly ASI
Name Group/Department	Aly lab, Beykoz Institute of Life Sciences and Biotechnology
Name Institute	Bezmialem Vakif University
City	Istanbul
Country	Turkey
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Name of the mutant parasite
RMgm number	RMgm-5366
Principal name	Pyaad(-)
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	Normal numbers of male and female gametocytes. However, male gamete formation (exflagellation) was completely abolished.
Fertilization and ookinete	No ookinete formation (in mosquito midguts)
Oocyst	No ookinete formation (in mosquito midguts); No oocyst formation
Sporozoite	No ookinete formation (in mosquito midguts); No oocyst formation; No sporozoite formation
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of AAD. Protein (function) A bacterial type amino acid decarboxylase (AAD), previously been annotated as UIS14 (up-regulated in infective sporozoites). A hallmark of this protein family is a conserved ornithine/arginine/lysine decarboxylase domain, which belongs to the group III pyridoxal-P-dependent a-amino acid decarboxylase family. This group encompasses prokaryotic ornithine, lysine and arginine carboxylases, which primarily function in amino acid catabolism and polyamine biosynthesis. See also RMgm-4078 for a P. berghei mutant lacking expression of AAD (with a somewhat different phenotype). Phenotype Normal growth/multiplication of asexual blood stages. Normal numbers of male and female gametocytes. However, male gamete formation (exflagellation) was completely abolished. No ookinete formation (in mosquito midguts); No oocyst formation; No sporozoite formation. See also RMgm-4078 for a P. berghei mutant lacking expression of AAD (with a somewhat different phenotype, i.e. reduced male gamete formation and normal ookinete formation and mosquito transmission). 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_1004800

Gene Model P. falciparum ortholog

PF3D7_0405700

Gene product

lysine decarboxylase, putative

Gene product: Alternative name

amino acid decarboxylase, AAD; UIS14

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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

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

Promoter of the selectable marker

unknown

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

For knockout, 5' and 3' UTR regions of PyAAD (PlasmoDB accession number PY17X_1004800) were amplified from genomic DNA of WT P. yoelii 17×-NL strain. For knock-in, about 800 bp of the open reading frame immediately upstream of the stop codon was amplified from genomic DNA. Right homologous regions (HR) were identical for both knockout and knock-in constructs. The amplified fragments were inserted into the transfection plasmid pAA20 between the SacII-BamHI, and HindIII-KpnI restriction enzyme recognition sites for knockout; and SacII-EcoRI, and HindIII-KpnI restriction enzyme recognition sites for knock-in.

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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