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Details of the target gene |
Gene Model of Rodent Parasite |
PBANKA_0412900
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Gene Model P. falciparum ortholog |
PF3D7_0315200
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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 seven tandem thrombospondin type I repeat-like (TS) domains |
Inducable system used | No |
Short description of the conditional mutagenesis | Not available |
Additional remarks inducable system |
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Type of plasmid/construct | Plasmid double cross-over |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
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Plasmid/construct map |
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Plasmid/construct sequence |
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Restriction sites to linearize plasmid |
NheI, SbfI
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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. 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 pCN-CTRP to obtain pCN-CTRP/DHR.
The six A domains were amplified with 5-CVWA/U (5’-GCGCGCCACAAATAAATTTATTATGTATTTCTACAGAAGA-3’) and 6-CVWA/D (5’-ACGCGTCACCATGTGCAGGT-3’), digested with MluI and BssHII, and cloned into MluI-digested pCN-CTRP/DHR to give pCAN-CTRP/DHR.
The plasmid backbone was excised with NheI and SbfI and and the linear construct was used for transfection. |
Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences 
Primer information: Primers used for amplification of the target sequences
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
Sequence Primer 3 | |
Additional information primer 3 | |
Sequence Primer 4 | |
Additional information primer 4 | |
Sequence Primer 5 | |
Additional information primer 5 | |
Sequence Primer 6 | |
Additional information primer 6 | |
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