Back to search resultsSummaryRMgm-843
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene mutation |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 23356439 |
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 SH |
Name Group/Department | Seattle Biomedical Research Institute |
Name Institute | Seattle Biomedical Research Institute |
City | Seattle |
Country | US |
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Name of the mutant parasite | |
RMgm number | RMgm-843 |
Principal name | Puf2-RBDmyc |
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 | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation Analysis of the mutant expressing the mutated form of Puf2 indicates that the RNA binding domain (RBD) is necessary and sufficient for all essential Puf2 functions, and parasites that express only the RBD retain normal infectivity and exhibit no phenotypic defects. |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_0719200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0417100 | ||||||||||||||||||||||||||
Gene product | mRNA-binding protein PUF2 | ||||||||||||||||||||||||||
Gene product: Alternative name | Pumilio2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | A puf2 gene lacking the N-terminal region but containing the RNA-binding domain with an 4xMyc tag | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | Plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
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 | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | Gene targeting constructs for transgenic parasite production were designed as previously described with some modifications (Mikolajczak et al., 2008a). Briefly, two regions of the targeted locus were PCR amplified. The two PCR products were fused by Sequence Overlap Extension PCR (“SOE PCR”) by virtue of complementary sequences designed into the primers. This SOE PCR product (“targeting sequence”) was digested at the 5’ and 3’ ends via exogenous restriction sites designed in the primers and inserted into a modified pDEF vector designed for genetic disruptions or for gene modifications by locus replacement via double-crossover recombination. Targeting sequences designed to express only the RNA-Binding Domain of Puf2 (“Puf2-RBDmyc”, AA132-478) were produced by an additional SOE PCR event to seamlessly fuse the promoter with the coding sequence without an intervening restriction enzyme site. Here, the correctly annotated wild-type locus was genetically modified by double-crossover recombination with a plasmid that seamlessly fused Puf2’s 5’UTR (with its start codon) to its RNA-Binding Domain (RBD) with an appended C-terminal 4xMyc tag (“Puf2-RBDmyc”) Prior to transfection, plasmids were linearized at unique restriction sites designed between the two halves of the targeting sequences | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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