SummaryRMgm-623
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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) : 21729699 |
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. Ramakrishnan; R.E. Sinden |
Name Group/Department | Division of Cell and Molecular Biology, Sir Alexander Fleming Building |
Name Institute | Imperial College London, South Kensington Campus |
City | London |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-623 |
Principal name | ΔA6 |
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 | Ookinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts. |
Oocyst | Ookinetes show reduced motility and invasion of midgut epithelium and do not develop into oocysts. |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0412900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0315200 | ||||||||||||||||||||||||||
Gene product | circumsporozoite- and TRAP-related protein | ||||||||||||||||||||||||||
Gene product: Alternative name | CTRP; circumsporozoite- and thrombospondin-related adhesive protein (TRAP)-related protein (CTRP) | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Mutated ctrp lacking the six A-domains | ||||||||||||||||||||||||||
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 | NheI, SbfI | ||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | A 682 bp fragment encompassing the 3’ untranslated region (UTR) and the 3’ region of ctrp was amplified from P. berghei genomic DNA using the primers 9-CTMCT3’/U (5’-GGTACCGGTAGTAGTCAGGAAATTCAAATGG-3’) and 10-CTMCT3’/D (5’-ACGCGTCGAATAATGGTGAAAATAGTGAAG-3’) and excised from an intermediate vector using MluI and KpnI digestion. The signal sequence of ctrp together with its 5’ UTR was amplified as a 2,158 bp fragment using 3-CPROSIG/U (5’- ACGCGTGCCTTCTTGATATATTTTTTTGAAG-3’) and 4-CPROSIG/D (5’-GAATTCGTGAAAATATGTGTATTTTTTCTTAAATTTG-3’) and was excised from an intermediate vector by EcoRI and MluI digestion. Both fragments were ligated into EcoRI-KpnI digested pDb.Dh.^ Db (de Koning-Ward et al., 1998), resulting in pCN-CTRP. All seven TS domains were then amplified with 7-CTSP/U (5'-ACGCGTCTAAAACGGGAGGTACACAAG-3') and 8-CTSP/D (5'-GCGCGCACCCCGAAGATGCAGAAT-3'), digested with MluI and BssHII, and cloned into MluI-digested pCN-CTRP to give pCTSN-CTRP. Finally, a 949 bp fragment of the ctrp 3’ UTR was amplified with the primers 17-C3’/U (5’-GGCCGTTGACTGAGGTAAAGGCATCCCTT-3’) and 18C3’/D (5’-GGCCGTTGACGAATGCTCATATGCGTGTG-3’), digested with HincII and cloned into HincII digested pCTSN-CTRP to obtain pCTSN-CTRP/DHR The plasmid backbone was excised with NheI and SbfI and and the linear construct was used for transfection. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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