SummaryRMgm-5533
|
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) : 39255311 |
MR4 number | |
top of page | |
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). |
top of page | |
The mutant parasite was generated by | |
Name PI/Researcher | Zeeshan M, Tewari T |
Name Group/Department | University of Nottingham |
Name Institute | School of Life Sciences |
City | Nottingham |
Country | UK |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-5533 |
Principal name | NEK1-GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
top of page | |
Phenotype | |
Asexual blood stage | NEK1-GFP was undetectable in ring stages but was visible with a diffuse cytoplasmic location during later stages (trophozoites). At the start of schizogony, NEK1-GFP re-located In the cytoplasm initially to single focal points near individual nuclei, which later divided into two tightly associated punctae. NEK1-GFP signal disappeared at the end of nuclear division and was undetectable in free merozoites. |
Gametocyte/Gamete | NEK1-GFP associates with MTOC formation and spindle dynamics during male gamete formation |
Fertilization and ookinete | Not tested |
Oocyst | The location of NEK1-GFP was also studied during other asexual stages like liver schizogony and mosquito gut sporogony. The live cell images of the proliferative liver schizogony and sporogony stages showed similar patterns of NEK1-GFP foci formation during proliferative stages. |
Sporozoite | Not tested |
Liver stage | The location of NEK1-GFP was also studied during other asexual stages like liver schizogony and mosquito gut sporogony. The live cell images of the proliferative liver schizogony and sporogony stages showed similar patterns of NEK1-GFP foci formation during proliferative stages. |
Additional remarks phenotype | Mutant/mutation
Generation of dual tagged parasite lines The analyses of these mutants revealed the following (from the Abstract): |
top of page | |||||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1443000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1228300 | ||||||||||||||||||||||||||
Gene product | NIMA related kinase 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | NEK1 | ||||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
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 | - GFP-tagging vectors were designed using the p277 plasmid vector. For GFP-tagging of NEK1 by single crossover homologous recombination, a region of nek1 downstream of the start codon was used to generate the construct. - NEK1 was tagged at the C-terminus with a 3xHA tag generated using PlasmoGEM vectors and a recombineering strategy (https://plasmogem.umu.se/pbgem/). - To study the function of NEK1, we used two conditional knock down systems: an auxin inducible degron (PP1AID) and a promoter exchange/trap using double homologous recombination (PP1PTD). The NEK1AID construct was derived from the p277 plasmid, where the GFP sequence was excised following digestion with AgeI and NotI restriction enzymes and replaced with an AID/HA coding sequence. The AID-HA sequence was PCR amplified (using primers: 5’-CCCCAGACGTCGGATCCAATGATGGGCAGTGTCGAGCT-3’ and 5’- ATATAAGTAAGAAAAACGGCTTAAGCGTAATCTGGA-3’) from the GW-AID/HA plasmid (http://plasmogem.sanger.ac.uk/). Fragments were assembled following the Gibson assembly protocol to generate the NEK1-AID/HA transfection plasmid that was transfected in the line. The conditional knockdown construct NEK1clag was derived from Pclag (pSS368) where nek1 was placed under the control of the clag promoter, as described previously (Sebastian et al., 2012). - Generation of dual tagged parasite lines The NEK1-GFP parasites were mixed with either NDC80-cherry or EB1-mCherry or ARK2-mCherry or kinesin-8B-mCherry parasites in equal numbers and injected into mice. Mosquitoes were fed on mice 4 to 5 days after infection when gametocyte parasitaemia was high. These mosquitoes were checked for oocyst development and sporozoite formation at day 14 and day 21 after feeding. Infected mosquitoes were then allowed to feed on naïve mice and after 4 - 746 5 days, and the mice were examined for blood stage parasitaemia by microscopy with Giemsa-stained blood smears. In this way, some parasites expressed both NEK1-GFP and NDC80-mCherry; or EB1-mCherry; or ARK2-mCherry; or kinesin-8B-mCherry in the resultant gametocytes, and these were purified, and fluorescence microscopy images were collected as described above. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
| |||||||||||||||||||||||||||
top of page |