SummaryRMgm-5272
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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) | Not published (yet) |
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 17X |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Goswami D, Vaughan AM |
Name Group/Department | Center for Global Infectious Disease Research |
Name Institute | Seattle Children’s Research Institute |
City | Seattle |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-5272 |
Principal name | Py linup‾ |
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 | Severe liver stage attenuation. in Pylinup− sporozoite infection with 1,000 sporozoites only one of ten mice became patent and this with delay (day twelve) and seven of twenty mice became patent when infection was done with 10,000 sporozoites with patency in the blood stage positive mice delayed to days seven through twelve. Five of ten mice became patent when using the 50,000 sporozoite dose and patency was delayed to days seven to nine. Highly susceptible BALB/cByJ mice infected with 50,000 wildtype sporozoites all became patent on day three after sporozoite infection, whereas only nine of twenty mice infected with Py linup− sporozoites became patent and the day to patency ranged from days eight to ten. Liver stage size measurements revealed that at 24 hours post-infection, Py linup− liver stages were comparable in size to wild-type. However, at both 36 and 48 hours post-infection, Py linup− liver stages were significantly smaller than wildtype with the size differences being more pronounced at the later time point post-infection. At 48 hours post infection, differences in protein expression and DNA replication/segregation were evident in Py linup− liver stages when compared to wildtype liver stages. Specifically, the branching of the mitochondria and apicoplast was reduced and DNA replication as well as DNA segregation appeared significantly reduced. Furthermore, the extensive invaginations of the liver stage plasma membrane that precedes merozoite formation called cytomere formation was severely disordered in Py linup− liver stages. This was evident from the changed expression pattern of the parasite plasma membrane marker MSP1, which localizes to the late liver stage schizont plasma membrane. |
Additional remarks phenotype | Mutant/mutation Phenotype Liver stage size measurements revealed that at 24 hours post-infection, Py linup− liver stages were comparable in size to wild-type. However, at both 36 and 48 hours post-infection, Py linup− liver stages were significantly smaller than wildtype with the size differences being more pronounced at the later time point post-infection. At 48 hours post infection, differences in protein expression and DNA replication/segregation were evident in Py linup− liver stages when compared to wildtype liver stages. Specifically, the branching of the mitochondria and apicoplast was reduced and DNA replication as well as DNA segregation appeared significantly reduced. Furthermore, the extensive invaginations of the liver stage plasma membrane that precedes merozoite formation called cytomere formation was severely disordered in Py linup− liver stages. This was evident from the changed expression pattern of the parasite plasma membrane marker MSP1, which localizes to the late liver stage schizont plasma membrane. Additional information |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1465200 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1249700 | ||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||
Gene product: Alternative name | LINUP, liver stage nuclear protein | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||
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 | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | Deletion of Py LINUP (PY17X_1465200) was achieved using CRISPR/Cas9 technology. In brief, LINUP was deleted using double crossover homologous recombination and complementary regions of LINUP upstream and downstream of the open reading frame were ligated into plasmid pYC L2 to create pYC_LINUP. pYC_LINUP was transfected into the blood stage schizonts of Py 17XNL. | ||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||
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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