RMgmDB - Rodent Malaria genetically modified Parasites
Back to search resultsSummaryRMgm-1155
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Last modified: 17 January 2015, 12:50
*RMgm-1155| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene mutation |
| Number of attempts to introduce the genetic modification | 4 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25565321 |
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| 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 | |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Ganter M, Matuschewski K |
| Name Group/Department | Parasitology Unit |
| Name Institute | Max Planck Institute for Infection Biology |
| City | Berlin |
| Country | Germany |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1243100 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0528500 | ||||||||||||||||||||||||||
| Gene product | F-actin-capping protein subunit alpha, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | CPalpha, CPα | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | P. berghei cpα replacement with a truncated (27bp C-terminal tail) P. falciparum cpα | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | (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 | |||||||||||||||||||||||||||
| 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 | Truncation of the 28 C-terminal amino acids of chicken CPα led to a 5000-fold reduction in capping affinity. Hence, a P. falciparum CPα replacement vector was generated containing the corresponding C-terminal truncation (PfCPα∆tail). Two independent transfections of this targeting plasmid, each conducted in duplicate, did not result in recombinant parasites. This result indicate that presence of the predicted actin-binding motif is essential for CPα function in vivo. See RMgm-1154 for a successful replacement of P. berghei cpα with the complete P. falciparum cpα gene. See RMgm-1153: Unsuccessful attempts to disrupt CPα indicating an essential function during blood stage growth/multiplication. In order to complement cpα(-) parasites, we amplified the orthologous P. falciparum CPα gene using the primers PfCPα_compforV and PfCPα_comprevVI and P. falciparum cDNA as template. Cloning into the plasmid pPbCPαrep resulted in the complementation plasmid pPfCPα. For complementation with a C-terminally deleted P. falciparum CPα gene that lacks the last 27 amino acid residues, primers PfCPα_compforV and PfCPα_comprevVII resulting in the complementation plasmid pPfCPα∆tail. See the link: http://www.pberghei.eu/index.php?t=4&cat=textterm&q=F-actin-capping%20protein&filter=all,disrupted,mutated,tagged,transgene,other&filter_transg=all,transgene,promoter,3utr For other mutants with mutated F-actin-capping protein subunits One of the few conserved actin-binding proteins of Plasmodium parasites is the F-actin capping protein (CP), which is found in all eukaryotic organisms and metazoan cell types CP binds in a calcium-independent manner to the fast growing (barbed) ends of F-actin, thereby blocking subunit exchange. CP also belongs to the defined set of proteins that are needed to reconstitute actin-based motility in vitro. Active CP is composed of two subunits, CPα and CPβ, and production of recombinant active CP in Escherichia coli (E. coli) is typically only achieved by co-expression of both subunits Plasmodium CPβ is encoded by a single open reading frame, whereas CPα is composed of nine small exons. Overall, Plasmodium CPα-subunits share approximately 19% amino acid sequence identity with other eukaryotic CPα-subunits, and 50-90% identity across different Plasmodium species. The residues that contribute to actin binding and heterodimer formation are conserved. CPβ-subunit of rodent malaria parasite P. berghei (PbCPβ) as an essential regulator of sporozoite motility and malaria transmission. Deletion of PbCPβ did not influence asexual and sexual blood-stage development in the mammalian host. In the insect vector, Anopheles mosquitoes, mutant parasites displayed defective motility, which completely arrested life cycle progression at the sporozoite stage. It has been shown that recombinant P. berghei CPα/β heterodimers display capping activity on heterologous non-muscle actin. The stage-specific function of CPβ in sporozoites implies that CPα alone might be functional during blood infection of cpβ(-) parasites. Given that independent functions of CP subunits have not been described, this notion was unexpected and prompted to investigate the cellular role(s) of Plasmodium CPα for parasite life cycle progression. In this study it is shown that the two CP subunits can be functionally separated. Unlike the beta subunit, the CPalpha subunit of the apicomplexan parasite Plasmodium is refractory to targeted gene deletion during blood infection in the mammalian host. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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