RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1103100; Gene model (P.falciparum): PF3D7_0503400; Gene product: actin-depolymerizing factor 1 (ADF1)
PhenotypeNo phenotype has been described
Last modified: 16 June 2010, 21:59
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 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 15975905
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherH. Schüler; K. Matuschewski
Name Group/DepartmentDepartment of Biochemistry and Biophysics
Name InstituteStockholm University

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1103100
Gene Model P. falciparum ortholog PF3D7_0503400
Gene productactin-depolymerizing factor 1
Gene product: Alternative nameADF1
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid XbaI
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe actin-depolymerizing factor (ADF)/cofilin family (AC proteins) are ubiquitous eukaryotic proteins that modulate the turnover of the microfilament system. ADF/cofilins bind F-actin in a 1:1 stoichiometry per actin subunit, an interaction that drastically destabilizes the polymers. The ADF/cofilin-induced acceleration of F-actin turnover is largely responsible for the rapid microfilament remodeling. AC proteins also bind monomeric actin (G-actin).
In this study two P. falciparum genes were identified with homology to ADF/cofilin genes (PfADF1, PFE0165w and PfADF2, PF13_0326, PBANKA_113750). Sequence comparisons showed that both Plasmodium ADFs share roughly 20–30% identity with yeast, plant, and animal ADF/cofilins. ADF1 lacks the F-actin binding residues of the AC consensus.
Evidence is presented that recombinant PfADF1 interacts with monomeric actin but does not bind to actin polymers.
The disruption of the PbADF1 locus was not successful in four independent transfection attempts.
To control for gene targeting at the desired locus, an integration control plasmid was included. After transfection the control plasmid results in a pseudodiploid allele with one functional PbADF1 copy. As expected, the latter resulted in viable recombinant parasites after a single transfection attempt.
Together these results indicate that ADF1 performs vital functions during asexual development of the malaria parasite.

The wild-type ADF1 genomic locus is targeted with a XbaI linearized targeting plasmid containing the positive selectable marker (dhfr/ts), A 5' truncation of the ADF1 open reading frame, and a 3' truncation of ADF1.

See RMgm-401 for a mutant lacking expression of ADF2 (PF13_0326; PBANKA_113750).
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
Additional information primer 1ADF1for (EcoRI)
Additional information primer 2ADF1rev1 (NotI)
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