RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1290

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1225400; Gene model (P.falciparum): PF3D7_0805200; Gene product: gamete release protein, putative \| IMC-associated apicomplexan protein, putative (GAMER; IAAP; PUA26 (protein unique to apicomplexan parasites, 26)) Name tag: GFP
Phenotype	Fertilization and ookinete;

Last modified: 11 June 2015, 21:17

*RMgm-1290

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) : 26018192
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	Kaneko I; Yuda M
Name Group/Department	Department of Medical Zoology
Name Institute	Mie University Graduate School of Medicine
City	Tsu, Mie
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-1290
Principal name	IAAP-GFP
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	GFP-tagged IAAP displayed a distribution pattern in mature ookinetes characteristic of SPN (subpellicular network) proteins (localization at the parasite surface, except at the apical end). By immunoelectron microscopy gold particles were localized predominantly along the parasite surface, except at the apical region. Under higher magnification, it was evident that the particles were localized not to the plasma membrane, but on the electron-dense structure beneath it, indicating that PUA26 is located on IMC (inner membrane complex).
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expressed a GFP-tagged version of GAMER/IAAP (gamete release protein, putative; IMC-associated apicomplexan protein, putative). The tagged gene is introduced on the centromere plasmid pCen-GFP (circular PAC). Protein (function) GAMER/IAAP encodes a small 96 amino acid protein (10.7 kD) lacking recognizable domains, as revealed by both manual annotation and three-dimensional (3D) homology modeling. Phenotype GFP-tagged IAAP displayed a distribution pattern in mature ookinetes characteristic of SPN (subpellicular network) proteins (localization at the parasite surface, except at the apical end). By immunoelectron microscopy gold particles were localized predominantly along the parasite surface, except at the apical region. Under higher magnification, it was evident that the particles were localized not to the plasma membrane, but on the electron-dense structure beneath it, indicating that IAAP is located on IMC (inner membrane complex). Two mutants have been published that lack expression of GAMER/IAAP. For these mutants a different phenotype has been reported. See RMgm-1289 and RMgm-1127 Additional information Other mutants RMgm-1289: A mutant lacking expression of GAMER/IAAP RMgm-1127: Another mutant lacking expression of GAMER/IAAP (with a different phenotype!!) RMgm-1228: A mutant expressing a GFP-tagged version of GAMER/IAAP

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1225400

Gene Model P. falciparum ortholog

PF3D7_0805200

Gene product

gamete release protein, putative | IMC-associated apicomplexan protein, putative

Gene product: Alternative name

GAMER; IAAP; PUA26 (protein unique to apicomplexan parasites, 26)

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

C-terminal

Additional remarks: tagging

To examine localisation of the protein, the ORF was expressed as a GFP-tagged protein under control of the original promoter, using the centromere plasmid pCen-GFP.

Commercial source of tag-antibodies

Type of plasmid/construct

Plasmodium Artificial Chromosome (PAC) circulair

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

tgdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Protein targeting was investigated by expressing the gene as a GFP-tagged protein, using the centromere plasmid pCen-GFP (circular PAC). Target genes with the upstream regulatory region (1.1–1.5-kbp upstream of the first methionine codon) were amplified by PCR using genomic DNA as the template, and then subcloned into the region immediately upstream of the GFP gene of the pCen vector.

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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