SummaryRMgm-788
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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) : 23028336 |
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 | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943) |
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The mutant parasite was generated by | |
Name PI/Researcher | D.S. Guttery; R. Tewari |
Name Group/Department | Centre for Genetics and Genomics, School of Biology, Queens Medical Centre |
Name Institute | University of Nottingham |
City | Nottingham |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-788 |
Principal name | PPKL-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 | The intensity of PPKL-GFP fluorescence in sexual stages was highest in the nuclear and cytoplasmic compartments of female gametocytes and zygotes, and in the apical cytoplasm of ookinetes. The protein was also detected in oocysts and sporozoites, but was not observed in activated microgametocytes or microgametes |
Fertilization and ookinete | The intensity of PPKL-GFP fluorescence in sexual stages was highest in the nuclear and cytoplasmic compartments of female gametocytes and zygotes, and in the apical cytoplasm of ookinetes. The protein was also detected in oocysts and sporozoites, but was not observed in activated microgametocytes or microgametes |
Oocyst | The intensity of PPKL-GFP fluorescence in sexual stages was highest in the nuclear and cytoplasmic compartments of female gametocytes and zygotes, and in the apical cytoplasm of ookinetes. The protein was also detected in oocysts and sporozoites, but was not observed in activated microgametocytes or microgametes |
Sporozoite | The intensity of PPKL-GFP fluorescence in sexual stages was highest in the nuclear and cytoplasmic compartments of female gametocytes and zygotes, and in the apical cytoplasm of ookinetes. The protein was also detected in oocysts and sporozoites, but was not observed in activated microgametocytes or microgametes |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype analyses of mutants lacking expression of PPKL (RMgm-686, RMgm-687) indicate an essential role of PPKL in maturation of ookinetes. Phenotype PPKL has protein phosphatase enzyme activity, and is highly expressed in female gametocytes and ookinetes. ppkl mRNA is also expressed in asexual blood stages, with the highest expression in schizonts relative to hsp70 and arginyl-tRNA synthetase genes used as controls. Analysis of a mutant expressing GFP-tagged PPKL shows that the intensity of PPKL-GFP fluorescence in sexual stages was highest in the nuclear and cytoplasmic compartments of female gametocytes and zygotes, and in the apical cytoplasm of ookinetes. The protein was also detected in oocysts and sporozoites, but was not observed in activated microgametocytes or microgametes Additional information |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1329500 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1466100 | ||||||||||||||||||||||||||
Gene product | protein phosphatase containing kelch-like domains | ||||||||||||||||||||||||||
Gene product: Alternative name | PPKL; PPalpha; PfPPα | ||||||||||||||||||||||||||
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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 | anti-GFP polyclonal antibody (Invitrogen) | ||||||||||||||||||||||||||
Type of plasmid/construct | Plasmid single 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 | BglII | ||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | For GFP-tagging by single homologous recombination, a 1047 bp region of ppkl starting 1693 bp downstream of the ATG start codon and omitting the stop codon was amplified using primers P1tag F (5'-CCCCGGTACCGAGCTCCGATAAAAATATATGGTGATATAC-3') and P1tag R (5'-CCCCGGGCCCTGGAGCCCCATAATTTAATTCTCTC-3'), producing an amplicon 1047 bp in length. This was inserted upstream of the gfp sequence in the p277 vector using KpnI and ApaI restriction sites. The p277 vector contains the human dhfr cassette, also conveying resistance to pyrimethamine. Before transfection, the sequence was linearized using BglII. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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