Summary

RMgm-3831
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1419700; Gene model (P.falciparum): PF3D7_1321400; Gene product: palmitoyltransferase DHHC8, putative
PhenotypeNo phenotype has been described
Last modified: 10 December 2016, 21:21
  *RMgm-3831
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Not published (yet)
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherPlasmoGEM (O. Billker; J. Rayner)
Name Group/DepartmentPlasmoGEM
Name InstituteWellcome Trust Sanger Institute
CityHinxton Cambridge
CountryUK

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1419700
Gene Model P. falciparum ortholog PF3D7_1321400
Gene productpalmitoyltransferase DHHC8, putative
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene is 'likely essential' for asexual blood stage growth/multiplication as determined by barcode PCR in a large pool of gene-deletion mutants.
P. berghei blood stages were transfected with a large pool of barcoded disruption (gene-deletion) vectors. These disruption vectors contained long modification arms to efficiently target the genes of interest. In addition the vectors contain gene-specific molecular barcodes. Co-transfecting multiple gene-deletion vectors in the same electroporation reproducibly generates complex pools of barcoded P. berghei mutants. Unsuccessful gene disruption/deletion or successful gene disruption/deletion is determined by the 'absence or presence' of the barcode in the population (as determined by barcode-specific PCR analysis).

This report is based on simultaneous phenotyping of mutants by barcode sequencing and is part of a large-scale genetic screen.

Asexual blood stage phenotype:
Growth rate phenotypes were obtained by counting barcodes on a next generation sequencer daily between days 4 and 8 post transfection. It is in the nature of the screen that genotypes of individual mutants were not validated.

* Essential: growth rate not significantly different from 0.1.
* Dispensable: growth rate not significantly different from 1 (corresponding to wild type).
* Slow: growth rate significantly above 0.1 but below 1 (p < 0.05).
* Fast: growth rate significantly above 1 (p<0.05).

The project website http://plasmogem.sanger.ac.uk/ has further information on methods and vector designs, provides tools for data visualisation and analysis, and allows researchers to request vectors to recreate mutants, confirm individual phenotypes and conduct more in-depth analyses.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6