RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5462

Malaria parasite	P. yoelii
Genotype
Tagged	Gene model (rodent): PY17X_0840300; Gene model (P.falciparum): Not available; Gene product: acyl-erythrocyte membrane associated protein 1CoA synthetase (EMAP1) Name tag: EGFP
Phenotype	Asexual bloodstage;

Last modified: 14 June 2024, 21:29

*RMgm-5462

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) : 37952150
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 17X
Name parent line/clone	cl1.1
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Siau A, Preiser PR
Name Group/Department	Nanyang Technological University, School of Biological Sciences
Name Institute	Nanyang Technological University, School of Biological Sciences
City	Singapore
Country	Singapore
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Name of the mutant parasite
RMgm number	RMgm-5462
Principal name	see gene model below
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	No
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Phenotype
Asexual blood stage	See below ('Additional remarks phenotype')
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal GFP tagged version of the gene shown below. The tagged gene is introduced as (an) additional copies (copy) on episomes under control of the eef1a promoter Protein (function) See additional information Phenotype Localization; GHOST + VESICLE + HCC In this study 44 P. yoelii proteins were C-terminal tagged with GFP and their localization in infected erythrocytes containing schizonts was determined by fluorescence microscopy. Four localization patterns could be distinguished by combining the microscopy pictures with the localization data available in the literature. The four localization patterns were: GHOST (red blood cell membrane), exported (HCC), periphery (PVM, PV or PPM), or internal (parasite). Additional information In this study 44 P. yoelii proteins were C-terminal tagged with GFP and their localization in infected erythrocytes containing schizonts was determined by fluorescence microscopy. The tagged gene is introduced as (an) additional copies (copy) on episomes under control of the eef1a promoter Four localization patterns could be distinguished by combining the microscopy pictures with the localization data available in the literature. The four localization patterns were: ghost (red blood cell membrane), exported (HCC), periphery (PVM, PV or PPM), or internal (parasite). 10 proteins predicted to be exported (into the spatial proteome) were examined using endogenous HA-tagging (C-terminal). Of the 10 rodent-specific proteins, PEXEL-positive PY17X_0701000, PY17X_0701100, and PY17X_1102300 are exported as predicted. The remaining seven (PY17X_1001800, PY17X_0526100, PY17X_1210300, PY17X_0802800, PY17X_0839600, PY17X_1321800, and PY17X_0701300) are effectively exported out of the parasite. Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_0840300

Gene Model P. falciparum ortholog

Not available

Gene product

acyl-erythrocyte membrane associated protein 1CoA synthetase

Gene product: Alternative name

EMAP1

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Details of the genetic modification

Name of the tag

EGFP

Details of tagging

C-terminal

Additional remarks: tagging

The mutant expresses a C-terminal GFP tagged version of the gene shown below. The tagged gene is introduced as (an) additional copies (copy) on episomes under control of the eef1a promoter

Commercial source of tag-antibodies

Type of plasmid/construct

Circular plasmid

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

The mutant expresses a GFP-tagged version of the protein. The transgene is introduced on a plasmid and is expressed under the control of the constitutive eef1a promoter of P. berghei.
Episomal overexpression plasmids for GFP-tagging were constructed using appropriate full-length sequences of the target genes amplified from P. yoelii genomic DNA or constructed by chemical gene synthesis (Integrated DNA Technologies). All fragments were then cloned into the AvrII/SmaI sites of the PYePL-GFP plasmid.

SLI knock-in construct for HA-tagging was generated by replacing the GFP tag of the T2A-ePL-GFP plasmid94 with a codon optimized 3xHA tag. Target homology arm fragments of the gene of interests (without stop codon) were amplified using KOD -Plus- Neo polymerase (Toyobo) from P. yoelii genomic DNA and inserted between the AvrII/ApaI sites of the modified PY-T2A-HA plasmid.

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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