RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-388
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Last modified: 16 June 2010, 21:59
*RMgm-388| Successful modification | The 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 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei NK65 |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | H. Schüler; K. Matuschewski |
| Name Group/Department | Department of Biochemistry and Biophysics |
| Name Institute | Stockholm University |
| City | Stockholm |
| Country | Sweden |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1103100 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0503400 | ||||||||||||||||||||||||
| Gene product | actin-depolymerizing factor 1 | ||||||||||||||||||||||||
| Gene product: Alternative name | ADF1 | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | 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 | XbaI | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Partial | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The 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 | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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