RMgmDB - Rodent Malaria genetically modified Parasites
Back to search resultsSummaryRMgm-1149
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*RMgm-1149| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 25438048 |
| 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 | Ferguson, DJP; Sinnis, P; Tewaria R |
| Name Group/Department | Centre for Genetics and Genomics |
| Name Institute | School of Life Sciences, Queens Medical Centre, University of Nottingham |
| City | Nottingham |
| Country | UK |
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| Name of the mutant parasite | |
| RMgm number | RMgm-1149 |
| Principal name | ΔNΔRep |
| 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 | Normal numbers of oocysts. In mosquitoes infected with ΔNΔRep parasites, no sporozoite development in oocysts could be observed by light microscopy at day 14 and no sporozoites were observed in homogenized midguts. |
| Sporozoite | No sporozoite formation in oocysts. |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation Its overall structure is highly conserved in all Plasmodium species, consisting of a central repeat region flanked by an NH2-terminal domain containing a conserved proteolytic cleavage site, and a COOH-terminal celladhesion domain, the thrombospondin repeat (TSR). Deletion of the csp gene gives rise to oocysts in which sporozoites do not develop, demonstrating a critical role for this protein in sporozoite development. Various studies have dissected the functional role of the NH2 and COOH-terminal regions during egress from oocysts, invasion of salivary glands, exit from the inoculation site and localization to and invasion of hepatocytes. After their release from oocysts, the NH2-terminus of CSP mediates adhesion to salivary glands and in the mammalian host, it masks the TSR, maintaining the sporozoite in a migratory state. Once in the liver, a regulated proteolytic cleavage event leads to the removal of the NH2-terminal third of the protein exposing the TSR an event that is critical for efficient invasion of hepatocytes by sporozoites. See the link PBANKA_040320 for other CSP related mutants |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0403200 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0304600 | ||||||||||||||||||||||||||
| Gene product | circumsporozoite (CS) protein | ||||||||||||||||||||||||||
| Gene product: Alternative name | CS; CSP | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | The endogenous cs gene with a cs gene lacking the repeat region and the NH2-terminus | ||||||||||||||||||||||||||
| 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 | The ΔNΔRep csp gene was generated using a PCR-based approach beginning with the previously published pCSRep DNfull plasmid in which the NH2-terminus after the signal sequence through to the end of Region I had been deleted. Primers were designed to flank the central repeat region in order to delete this region and include restriction sites to yield fragments that when ligated made the desired csp deletion mutant. A 722 bp fragment, from the KpnI site in the 5'UTR through the csp signal sequence, was amplified using forward primer P1 (5-AAAAAAGGTACCAAATATTATATGC-3''; existing KpnI site underlined) and reverse primer P2 (5'-AGAGCAGCTGTCCATATCCTGGAAGT AGAG-3'; introduced PvuII site underlined). A 321 bp csp fragment, from the end of the repeat region through the stop codon, was amplified using forward primer P3 (5'-CCACAGCCACCCGGGAATAACAATAAC-3'; introduced SmaI site underlined) and reverse primer P4 (5'-GTTTATTTAATTAAAGAATACTAATAC-3'; existing PacI site underlined). Both PCR products were gel purified using the QIAquick gel extraction kit (Qiagen) and then digested with the appropriate restriction enzymes and ligated overnight at 15°C using T4 DNA ligase. Because several ligation products were possible, we performed a PCR amplification to identify the correct ligation product with primers spanning the 1043 bp KpnI-PacI fragment. The DnDrep csp gene was then cloned into pCR4-TOPO and sequenced. The KpnI-PacI fragment was then cloned into the transfection vector, pCSRep, replacing WT csp. After initial transfections failed because the repeat portion of CSP was re-inserted by the parasite during homologous recombination, the pCSRep vector was altered to include a longer, 1.5 kb 3'UTR. Since there was already 450 bp of 3'UTR in the transfection vector, we amplified from P. berghei ANKA gDNA, an additional 1235 bp fragment downstream of this using forward primer P5 (5'-ACAATATTATTTAAGGGAATTCTAA-3'; existing EcoRI site underlined) and reverse primer P6 (5'- CAGTGGAATTCTGAACTACCTG-3'; introduced EcoRI site underlined). The 1235 bp PCR product was gel purified and digested with the appropriate restriction enzymes before ligating into pCSRep which has a drug selection cassette consisting of the human DHFR gene flanked by the 5' and 3' UTRs of P. berghei DHFR-TS, followed by a csp cassette. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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