RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-208

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0905900; Gene model (P.falciparum): PF3D7_1143100; Gene product: AP2 domain transcription factor AP2-O, putative (AP2-O, AP2 in ookinetes; ApiAP2) Name tag: GFP
Phenotype	Fertilization and ookinete;

Last modified: 14 June 2010, 10:46

*RMgm-208

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) : 19220746
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	M. Yuda, I. Kaneko
Name Group/Department	Department of Medical Zoology
Name Institute	Mie University School of Medicine
City	Mie
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-208
Principal name	AP2-O::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not different from wild type
Fertilization and ookinete	No expression was observed in the gametocyte stage. Weak GFP fluorescent signals were observed in retort-form ookinetes beginning 8 h after fertilization. The signals were localized in the nucleus and signal intensities increased with ookinete development.
Oocyst	Not tested
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant expresses a GFP-tagged form of a transcription factor with AP2 domain(s) (AP2-O, AP2 in ookinetes) Protein (function) AP2-O belongs to the Apetala2 (AP2) family which encode transcription factors (TFs). The AP2 family TFs were first identified in plants. Plant AP2 family TFs are named for their possession of at least one common DNA-binding domain of approximately 60 amino acids, the AP2 domain. Bioinformatic analyses have revealed the presence of 26 AP2-related genes in the Plasmodium genome. The predicted Plasmodium AP2-related genes encode proteins with one to four AP2 domains. AP2-O has two conserved regions which include the AP2 domain near the C-terminus. See also mutant RMgm-207 lacking expression of AP2-O. Phenotype analyses of this mutant indicate an essential role of AP2-O during ookinete development and indicate that AP2-O is a transcription factor that binds to promoter regions of genes that are expressed during ookinete development and activates expression of genes, including genes involved in mosquito midgut invasion. Phenotype In wild type parasites ap2-0 transcripts are present in both gametocytes and ookinetes. No GFP expression was observed in the gametocyte stage indicating translational repression of the ap2-o transcripts in gametocytes. Weak GFP fluorescent signals were observed in retort-form ookinetes, beginning at 8h after fertilization. The signals were localized in the nucleus and signal intensities increased with ookinete development. See also mutant RMgm-207 lacking expression of AP2-O. Additional information Other mutants RMgm-207: A mutant lacking expression of AP2-O. RMgm-399: a mutant lacking expression of AP2-Sp (AP2 in sporozoites; PBANKA_132980;PF14_0633) RMgm-398: a mutant expressing a GFP-tagged form of AP2-Sp (PBANKA_132980;PF14_0633) RMgm-400: a mutant expressing a mutated form of AP2-O (PBANKA_090590; PF11_0442) whose AP2 domain had been swapped with that of AP2-Sp (PBANKA_132980;PF14_0633)

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0905900

Gene Model P. falciparum ortholog

PF3D7_1143100

Gene product

AP2 domain transcription factor AP2-O, putative

Gene product: Alternative name

AP2-O, AP2 in ookinetes; ApiAP2

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

C-terminal

Additional remarks: tagging

To construct AP2-O::GFP parasites, the targeting vector containing a marker gene, human DHFR (hDHFR), was integrated into the AP2-O locus by double-cross-over recombination, adding the GFP gene to the C-terminal portion of AP2-O and conferring pyrimethamine resistance to the AP2-O::GFP parasites

Commercial source of tag-antibodies

Type of plasmid/construct

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

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The targeted insertion vector was constructed in a pBluescript plasmid (Stratagene, La Jolla, CA, USA). For the targeted insertion construct, a DNA fragment containing the 3' part of the AP2-O coding region was amplified by PCR and inserted into the pBluescript XhoI/NheI site in frame with the GFP gene. The downstream region of the AP2-O gene was also amplified by PCR and inserted into the pBluescript BamHI/NotI site. Plasmids containing the construct were separated from plasmids without the construct by digestion with XhoI and NotI. AP2-O::GFP parasites were obtained by inserting the construct into the AP2-O locus by homologous recombination.

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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