Back to search resultsSummaryRMgm-4636
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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) : 31126959 |
MR4 number | |
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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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The mutant parasite was generated by | |
Name PI/Researcher | Spreng B, Frischknecht F |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | Heidelberg University Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-4636 |
Principal name | a1-tubulin(-) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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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 | Both a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line. Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding. |
Sporozoite | Both a1-tubulin(-) parasite lines showed no defect in blood-stage growth or initial mosquito infection. However, no sporozoites were found in the midgut or salivary glands of the mosquito; thus, a main defect seems to occur at the oocyst stage. This defect could be completely rescued by complementing the gene in the a1-tubulin(-) parasite line. Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding. |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phenotype - Deletion of a1-tubulin affects Plasmodium sporozoite formation during budding. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0417700 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0903700 | ||||||||||||||||||||||||
Gene product | alpha tubulin 1 | ||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||
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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 | Plasmids were designed for double-homologous crossover integration into the Plasmodium genome. The Pb262 vector (Singer et al, 2015) was modified by replacing the chromosome 12 integration sites with a1-tubulin 5UTR and 3'UTR integration sites for all vectors used in this study. Furthermore, the mCherry open reading frame (ORF) and the dhfs 30UTR were replaced with a set of different versions of a1-tubulin or a1/a2-tubulin chimeras and the a1-tubulin 3'UTR, respectively. In the case of the a2+++ vector, the dhfs 30UTR was replaced by a a2-tubulin 3'UTR. In case of the a1-tubulin(-) vector, the mCherry and the dhfs 3'UTR were deleted. All vectors contained the positive–negative selection marker hdhfr-yfcu and could be used for either the “gene insertion/marker out” technique (Lin et al, 2011) or the standard transfection protocols (Janse et al, 2006a). Final vectors were linearized before transfection by SalI and KpnI in case of the GIMO technique or by SalI and XhoI (or ScaI) for standard transfection. The linearized a1-tubulin(-) vector was transfected into an unmodified P. berghei strain ANKA or P. berghei strain ANKA expressing mCherry under the CSP and eGFP under the ef1a promoter using standard protocols. Parasites that integrated the desired DNA construct were selected by administration of pyrimethamine (0.07 mg/ml) via the mouse drinking water. An isogenic population was obtained by a dilution series, which was then followed by elimination of the positive–negative selection marker hdhfr-yfcu by applying 5-fluorocytosine (1 mg/ml). Another dilution series was performed to obtain an isogenic population of the selection marker-free a1-tubulin(-) RG parasites, which were used for complementation approaches with WT (a1a1(-) compl), deletion of a1-tubulin introns (a1Δintrons) and a set of a1/a2-tubulin chimera constructs (a2+++, a2++, a2+). The a1-tubulin codon-modified and intron-deleted construct (a1 cm&Δintrons) was transfected into the P. berghei strain ANKA. A receiver parasite line only differing in the dhfs 30UTR was used to generate the C-terminally truncated a1-tubulin parasite line (a1Δc-term) via a “gene insertion/marker out” approach. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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