SummaryRMgm-4749
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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) : 32049018 |
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 | Pandey R, Tewari R |
Name Group/Department | School of Life Sciences, Queens Medical Centre |
Name Institute | University of Nottingham |
City | Nottingham |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-4749 |
Principal name | SMC2mCherry |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | We found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. In early schizonts within host red blood cells, we observed discrete foci in the parasite cell adjacent to the nuclear DNA for both SMC2 and SMC4, whereas in mature schizonts, the signal was dispersed throughout the nucleus. To examine the colocalization of SMC2 and SMC4 proteins, we generated transgenic parasite lines expressing either SMC2mCherry or SMC4GFP and crossed them genetically. The progeny, expressing both SMC2mCherry and SMC4GFP, showed colocalization of the two proteins during schizogony and gametogenesis consistent with SMC2 and SMC4 heterodimer complex formation at both stages. |
Gametocyte/Gamete | We found that SMC2 and SMC4 are expressed during both schizogony and male gametogenesis. During male gametogenesis, the proteins were also dispersed throughout the nucleus. To validate the SMC4 subcellular location, fractionation of cytoplasmic and nuclear extracts derived from purified gametocytes revealed the presence of SMC4 in the nucleus. To examine the colocalization of SMC2 and SMC4 proteins, we generated transgenic parasite lines expressing either SMC2mCherry or SMC4GFP and crossed them genetically. The progeny, expressing both SMC2mCherry and SMC4GFP, showed colocalization of the two proteins during schizogony and gametogenesis consistent with SMC2 and SMC4 heterodimer complex formation at both stages. |
Fertilization and ookinete | In addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages |
Oocyst | In addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages |
Sporozoite | In addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages |
Liver stage | In addition, we observed SMC4GFP distributed either as dispersed in the nucleus or at a discrete focus adjacent to the DNA throughout the parasite life cycle, including in female gametocytes, in ookinetes, during oocyst development, and in the liver stages |
Additional remarks phenotype | Mutant/mutation Phenotype To examine the colocalization of SMC2 and SMC4 proteins, we generated transgenic parasite lines expressing either SMC2mCherry or SMC4GFP and crossed them genetically. The progeny, expressing both SMC2mCherry and SMC4GFP, showed colocalization of the two proteins during schizogony and gametogenesis consistent with SMC2 and SMC4 heterodimer complex formation at both stages. From the Abstract |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1416900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1318400 | ||||||||||||||||||||||||||
Gene product | structural maintenance of chromosomes protein 2, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | SMC2 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | mCherry | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) 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 | |||||||||||||||||||||||||||
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 | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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