RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1291

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1312700; Gene model (P.falciparum): PF3D7_1449000; Gene product: gamete egress and sporozoite traversal protein, putative (GEST) Name tag: GFP
Phenotype	Fertilization and ookinete;

Last modified: 11 June 2015, 21:58

*RMgm-1291

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-1291
Principal name	GEST-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 GEST localized in micronemes of ookinetes.
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expressed a GFP-tagged version of GEST (gamete egress and sporozoite traversal protein, putative). The tagged gene is introduced on the centromere plasmid pCen-GFP (circular PAC). Protein (function) By analysis of a mutant (RMgm-667) lacking expression of GEST evidence has been presented that male and female gametes fail to egress from the erythrocyte. Fertilisation, ookinete and oocyst production is strongly reduced. Phenotype GFP-tagged GEST localized in micronemes of ookinetes. Additional information In different studies GEST has been localized in osmiophilic bodies of gametocytes, micronemes of ookinetes and in sporozoites. It has been suggested that this protein plays a role both in egress (gametocytes) and in invasion of host cells (ookinetes, sporozoites) Other mutants RMgm-667: A mutant lacking expression of GEST RMgm-668: A mutant expressing a C-terminal GFP-tagged form of GEST

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1312700

Gene Model P. falciparum ortholog

PF3D7_1449000

Gene product

gamete egress and sporozoite traversal protein, putative

Gene product: Alternative name

GEST

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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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