RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4723

Malaria parasite	P. yoelii
Genotype
Tagged	Gene model (rodent): PY17X_0507300; Gene model (P.falciparum): PF3D7_1022000; Gene product: RNA-binding protein, putative (UIS12) Name tag: GFP
Phenotype	Sporozoite; Liver stage;

Last modified: 6 February 2020, 13:04

*RMgm-4723

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) : 31673027
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	Lindner, SE, Kappe SHI
Name Group/Department	Department of Global Health
Name Institute	University of Washington
City	Seattle
Country	US
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Name of the mutant parasite
RMgm number	RMgm-4723
Principal name	UIS12::-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	Not tested
Oocyst	Not tested
Sporozoite	PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Using live fluorescence microscopy with PyWT-GFP or PyUIS12::GFP sporozoites, we clearly observed GFP expression in control P. yoelii WT-GFP sporozoites, but did not detect UIS12::GFP protein when transcribed from its native locus. In agreement with translational repression being relieved posttransmission, IFA micrographs clearly show UIS12::GFP expression in the cytosol of 24-h old liver stage parasites.
Liver stage	PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Using live fluorescence microscopy with PyWT-GFP or PyUIS12::GFP sporozoites, we clearly observed GFP expression in control P. yoelii WT-GFP sporozoites, but did not detect UIS12::GFP protein when transcribed from its native locus. In agreement with translational repression being relieved posttransmission, IFA micrographs clearly show UIS12::GFP expression in the cytosol of 24-h old liver stage parasites.
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal GFP-tagged version of UIS12 Protein (function) PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Phenotype PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Using live fluorescence microscopy with PyWT-GFP or PyUIS12::GFP sporozoites, we clearly observed GFP expression in control P. yoelii WT-GFP sporozoites, but did not detect UIS12::GFP protein when transcribed from its native locus. In agreement with translational repression being relieved posttransmission, IFA micrographs clearly show UIS12::GFP expression in the cytosol of 24-h old liver stage parasites. Additional information Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_0507300

Gene Model P. falciparum ortholog

PF3D7_1022000

Gene product

RNA-binding protein, putative

Gene product: Alternative name

UIS12

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) plasmid single cross-over

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

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Reverse genetic modification of P. yoelii parasites. Plasmodium yoelii (17XNL strain) was genetically modified using conventional, double homologous recombination approaches with the pDEF plasmid vector. The 3′ end of py17X_1354300 or pyuis12 (PY17X_0507300) was modified by the addition of the GFPmut2 coding sequence prior to the stop codon. Transgenic parasites were identified by genotyping PCR, with independent transgenic clones being isolated by limiting dilution cloning. Clonal parasites were transmitted to A. stephensi mosquitoes to produce salivary gland sporozoites.

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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