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Details of the target gene |
Gene Model of Rodent Parasite |
PY17X_1114100
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Gene Model P. falciparum ortholog |
PF3D7_0513300
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Gene product | purine nucleoside phosphorylase |
Gene product: Alternative name | PNP |
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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 used | 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 |
BseRI
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Partial or complete disruption of the gene | Complete |
Additional remarks partial/complete disruption |
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Selectable marker used to select the mutant parasite | hdhfr |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | For deletion of pnp locus by replacement, a PCR-based approach for generation of gene targeting constructs for P. berghei was adapted for P. yoelii. Two PCR reactions: PCRI and PCRII, required to generate the complete constructs, were carried out by using the Advantage 2 Polymerase Mix (BD Biosciences) and Long Template PCR Polymerase Mix (Roche), respectively, according to the manufacturer’s instructions. PCR I amplified two flanking fragments of homology to the pnp gene from P. yoelii genomic DNA using primer combination of Pypnp-5’-F and Pypnp-5’-R or primer combination of Pypnp-5’-F and PypnpGFP-5’-R for the 5’-upstream region and primer combination of Pypnp-3’-F and Pypnp-3’-R or primer combination of PypnpGFP-3’-F and Pypnp-3’-R for the 3’-downstream region of pnp gene. Primers Pypnp-5’-R and Pypnp-3’-F have 5’-terminal extensions homologous to the selection cassette hdhfr of pL006 (kindly provided by Andy Waters and Chris Janse, Leiden); primers PypnpGFP-5’-R and PypnpGFP-3’-F have 5’ terminal extensions homologous to the selection cassette hdhfr/gfp of pHDGFP. For PCR II, the pnp 5’ and 3’ flanking fragments (1.5 kb each) with corresponding extensions from PCR I were combined with either ScalI-linearized pL006 or ScalI- linearized pHDGFP with the outer primers 5’-Pypnp-F and 3’-Pypnp-R. This leads to the amplification of the complete gene targeting constructs for deletion of pnp |
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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