SummaryRMgm-4649
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 31231888 |
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 | Yuda M, Kato T |
Name Group/Department | Department of Medical Zoology |
Name Institute | Mie University School of Medicine |
City | Tsu, Mie |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-4649 |
Principal name | AP2-FG(−) |
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 | Normal production of male gametocytes (and exflagellation). No production of mature female gametocytes. Evidence provided that immature female gametocytes are produced. |
Fertilization and ookinete | Strongly reduced numbers of ookinetes in in vitro cultures. All ookinetes show aberrant morphology. |
Oocyst | No oocyst production |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation See also other mutants in the databse targeting this gene PF3D7_1317200 Both clones proliferated at rates similar to wild-type parasites and produced mature male gametocytes with normal exflagellation. However, no mature female gametocytes were observed in them; instead, immature female gametocyte-like parasites (i.e., small mononuclear cells with a light violet cytoplasm) were observed on Giemsa-stained blood smears in both clones. Because it was difficult to conclude that they were female gametocytes, we further transfected AP2-FG(−)1 parasites with a centromere plasmid that conferred female-specific expression of the red fluorescent protein mCherry. Here, the small mononuclear parasites displayed red fluorescence, indicating that they were indeed female gametocytes. By flow cytometric analysis, the rate of mCherry-positive cells in AP2-FG(−) parasites was shown to be similar to that in wild-type parasites transfected with the same centromere plasmid, but the fluorescence intensity was significantly weaker than that in the wild-type. In contrast, when AP2-FG(−)1 parasites were transfected with a centromere plasmid that conferred the male-specific expression of mCherry, the rates of mCherry-positive cells and signal intensities were essentially the same as those of wild-type parasites. Collectively, these results suggested that female gametocytes were produced in AP2-FG(−) parasites, but they were unable to develop properly, and the expression of female-specific genes might decrease significantly. From the Abstract: |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1415700 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1317200 | ||||||||||||||||||||||||
Gene product | AP2 domain transcription factor AP2-FG, putative | ||||||||||||||||||||||||
Gene product: Alternative name | AP2-FG, ApiAP2 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) 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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Partial | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 | For the disruption, a construct containing the pyrimethamine-resistant selectable marker gene was inserted into the 5′ side of the coding region of the AP2 domain by double cross-over homologous recombination. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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