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Type and details of transgene |
Is the transgene Plasmodium derived |
Transgene: not Plasmodium |
Transgene name | RFP |
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Details of the genetic modification |
Inducable system used | No |
Additional remarks inducable system |
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Type of plasmid/construct | (Linear) 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 |
SbfI
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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 | A two step PCR technique was adapted for the efficient generation of the targeting construct. See figure 1: Mikolajczak et.al., Mol Biochem Parasitol 158 (2007) 213-216. In this paper a cloning procedure is described for gene replacement by double homologous recombination in P. yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the mutant parasites, a fluorescent protein (RFP) cassette is introduced into the targeting vector in which RFP is under the control of the constitutive eef1a promoter. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker. |
Additional remarks selection procedure | |
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Other details transgene |
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Promoter |
Gene Model of Parasite |
PBANKA_1133300
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Gene Model P. falciparum ortholog |
PF3D7_1357100
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Gene product | elongation factor 1-alpha |
Gene product: Alternative name | eef1a |
Primer information details of the primers used for amplification of the promoter sequence
Primer information details of the primers used for amplification of the promoter sequence
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
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3'-UTR |
Gene Model of Parasite |
PBANKA_0719300
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Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative |
Gene product: Alternative name | dhfr/ts |
Primer information details of the primers used for amplification the 3'-UTR sequences
Primer information details of the primers used for amplification the 3'-UTR sequences
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
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Insertion/Replacement locus |
Replacement / Insertion | Replacement locus |
Gene Model of Parasite |
PY00204
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Gene product | Not available |
Gene product: Alternative name | UIS4, up-regulated in infective sporozoites gene 4 |
Primer information details of the primers used for amplification of the target sequences
Primer information details of the primers used for amplification of the target sequences
Sequence Primer 1 | GCgatatcTTACAGATGATCAGAAGGAA |
Additional information primer 1 | Pr.1 (EcoRV); 3'UTR forward + amplification of the final PCR product |
Sequence Primer 2 | TAAATAAATGcctgcaggCAAAAATAATACACACTCTCAC |
Additional information primer 2 | Pr.2 (SbfI); 3'UTR reverse + annealing of 5'UTR and 3'UTR |
Sequence Primer 3 | TTATTTTTGcctgcaggCATTTATTTATTTTCCCTATT |
Additional information primer 3 | Pr.3 (SbfI); 5'UTR forward + annealing of 5'UTR and 3'UTR |
Sequence Primer 4 | GCgcggccgcATAAAAGGAAAGGATCAACC |
Additional information primer 4 | Pr.4 (NotI); 5'UTR reverse + amplification of the final PCR product |
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