SummaryRMgm-5203
|
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) : 35427579 |
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 | Not applicable |
Other information parent line | |
top of page | |
The mutant parasite was generated by | |
Name PI/Researcher | Srivastava PN, Mishra S |
Name Group/Department | Division of Molecular Microbiology and Immunology |
Name Institute | CSIR-Central Drug Research Institute |
City | Lucknow |
Country | India |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-5203 |
Principal name | Pla1- |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
top of page | |
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 | Salivary gland sporozoite in vivo infectivity was checked by i.v injecting in C57BL/6 mice. Initially, 5,000 sporozoites from WT GFP or Pla1- were used, and while the appearance of blood-stage infection was observed on the third day in all mice infected with WT GFP, a delay of 2.5 – 3.5 days was observed in the appearance of Pla1- blood-stage infection. To quantify the sporozoite invasion and subsequent development in the liver, we infected mice with 5x103 sporozoites and the liver parasite burden was quantified at 40 h p.i. by measuring the parasite 18S rRNA copy number using real-time PCR. There was no significant difference in liver parasite burdens between WT GFP and Pla1-. EEFs were also counted at 40 h p.i. in multiple experiments and no difference from wild type was observed, signifying normal productive invasion and development by Pla1- sporozoites (Fig. 4C). EEF growth was also monitored by measuring their areas at 40 h and 65 h p.i., with no significant difference from WT GFP. Subsequently, maturation of EEFs and the formation of merozoites was observed by anti-MSP1 staining. Both nuclear division and merozoite formation appeared normal in Pla1-EEFs. In vitro cell detachment was quantified for Pla1- and comparatively fewer detached cells (DCs) were detected floating in the cell culture medium even up to 70 h p.i. Evidence is presented that Pla1 has a role in merosome release. |
Additional remarks phenotype | Mutant/mutation Phenotype Additional information |
top of page | |||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1423100 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0814400 | ||||||||||||||||||||||||
Gene product | putative phospholipase, DDHD1 | ||||||||||||||||||||||||
Gene product: Alternative name | PA-PLA1, Pla1 | ||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||
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 | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 | For deleting PbPla1, two homology regions were amplified from the 5’ and 3’ untranslated regions (UTRs) of the gene using primer pairs 1147/1148 (F1) and 1149/1150 (F2) and cloned at XhoI/SalI (F1) and NotI/AscI (F2), respectively, in the pBC-GFP-hDHFR:yFCU vector. The final construct was linearized by XhoI/AscI digestion | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
![]() Primer information: Primers used for amplification of the target sequences
![]()
| |||||||||||||||||||||||||
top of page |