RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4426

Malaria parasite	P. yoelii
Genotype
Tagged	Gene model (rodent): PY17X_1121600; Gene model (P.falciparum): PF3D7_0621300; Gene product: mRNA-binding protein PUF3 (PUF3) Name tag: GFP
Phenotype	Asexual bloodstage; Oocyst; Sporozoite; Liver stage;

Last modified: 7 March 2018, 18:02

*RMgm-4426

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) : 29487181
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	Liang X, Lindner SE, Cui L
Name Group/Department	Department of Entomology
Name Institute	Pennsylvania State University
City	University Park
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-4426
Principal name	PyPuf3::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Nuclear, nucleolar location of GFP-tagged Puf3 in blood stages.
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Puf3 changed its localization in mosquito and liver stages from nucleolar to cytosolic puncta. After transmission of the transgenic parasite line to mosquitoes, PyPuf3::GFP was observed to be concentrated into banded foci in the developing oocysts. When sporozoites were dissected from the developing oocysts on day 10, PyPuf3::GFP was found in punctate foci of the oocyst sporozoites. The PyPuf3::GFP foci were not observed in mature salivary gland sporozoites.
Sporozoite	Puf3 changed its localization in mosquito and liver stages from nucleolar to cytosolic puncta. After transmission of the transgenic parasite line to mosquitoes, PyPuf3::GFP was observed to be concentrated into banded foci in the developing oocysts. When sporozoites were dissected from the developing oocysts on day 10, PyPuf3::GFP was found in punctate foci of the oocyst sporozoites. The PyPuf3::GFP foci were not observed in mature salivary gland sporozoites.
Liver stage	Puf3 changed its localization in mosquito and liver stages from nucleolar to cytosolic puncta. During mid-liver stage development (24 h), large foci containing PyPuf3::GFP were visible, and they were adjacent to DAPI stained DNA but had minimal overlap. However, in late (48 h) liver stage schizonts, PyPuf3::GFP expression was not detectable.
Additional remarks phenotype	Mutant/mutation The mutant expresses a GFP-tagged version of Puf3 Protein (function) See additional information. Phenotype Nuclear, nucleolar location of GFP-tagged Puf3 in blood stages. Puf3 changed its localization in mosquito and liver stages from nucleolar to cytosolic puncta. Additional information In the paper evidence is presented that: - Secondary structure prediction suggested that the RNA-binding domains of Puf3s consisted of 11 pumilio repeats, similar to those in human Puf-A and yeast Puf6, which are involved in ribosome biogenesis. - Neither pfpuf3 nor pypuf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle - Nuclear, nucleolar location - PyPuf3 changed its localization in mosquito and liver stages from nucleolar to cytosolic puncta. - Affinity purification of PTP-tagged variant of PfPuf3 revealed an association with 31 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together, these results suggest an essential function of PfPuf3 in ribosomal biogenesis Other mutants

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_1121600

Gene Model P. falciparum ortholog

PF3D7_0621300

Gene product

mRNA-binding protein PUF3

Gene product: Alternative name

PUF3

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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 double 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

unknown

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

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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