SummaryRMgm-770
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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) : 22393411 |
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 | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943) |
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The mutant parasite was generated by | |
Name PI/Researcher | C. Aldrich; R. Spaccapelo |
Name Group/Department | Department of Experimental Medicine |
Name Institute | University of Perugia |
City | Perugia |
Country | Italy |
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Name of the mutant parasite | |
RMgm number | RMgm-770 |
Principal name | PgCS/PbRR clones 4 and 5 |
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 | Not different from wild type |
Sporozoite | Normal numbers of oocysts and midgut sporozoites (at day 14 post infection). Strongly reduced numbers of salivary gland sporozoites at day 21 p.i. Salivary gland sporozoites are unable to infect mice after intravenous inoculation |
Liver stage | Salivary gland sporozoites are unable to infect mice after intravenous inoculation |
Additional remarks phenotype | Mutant/mutation Additional information Figure 1 |
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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 | Endogenous csp replaced by csp of P. galinaceum with the repeat region of P. bergehi csp | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | 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 | ApaI | ||||||||||||||||||||||||||
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 targeting construct pPgCS/PbRR, generated in this study contains the following structural elements: (i) the first 1,130 nucleotides of the PbCS 5’UTR sequence immediately upstream of the start codon; (ii) a chimeric form of the CS coding sequence and (iii) the first 1,150 nucleotides of the PbCS 3’ UTR sequence immediately downstream of its stop codon, in which the T. gondii dihydrofolate reductase/thymidylate synthase (TgDHFR/TS) drug selectable marker cassette (5,150 bp) was inserted at its HindIII site. The constructs was generated from pPgCSSX (RMgm-769) by replacing the PgCS repeat region in pPgCSSX with the PbCS repeat region. The 1.02 kb PbCS gene was amplified from P. berghei genomic DNA (Anka strain, clone 2.34) and sequenced. Forward and reverse primers were designed to amplify the PbCS repeat region from this sequence, introducing the SpeI and XhoI sites at either end of the repeat region (Spefor 5’-TAATAATAAATTGAAACAACCAACTAGTCCACCACCACCAAACCCAAATG- 3’ (SpeI is underlined) and XhoRev 5’-CCGCTCGAGGATATAAGAATCGTCATTATTATTATTTTTGTTATTG- 3’ (XhoI is underlined)). The PgCS repeat region was then replaced with the PbCS repeat region, via SpeI and XhoI digestions. The targeting construct was designed to direct the double cross-over event between the 1.13 kb sequence of the PbCS 5’ untranslated region (UTR) and the 0.85 kb sequence of the PbCS 3’ UTR in the linearized plasmid and their corresponding sequences in the PbCS locus. Plasmids pPgCSSX was digested with ApaI to release the 8.6 kb targeting insert from the plasmid backbone. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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