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Details of the target gene |
Gene Model of Rodent Parasite |
PBANKA_0712900
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Gene Model P. falciparum ortholog |
PF3D7_0817900
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Gene product | high mobility group protein B2 |
Gene product: Alternative name | HMGB2 |
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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 | (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 |
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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 | Two PCR fragments flanking the HMGB2 open reading frame (ORF) were amplified from the genomic DNA using oligonucleotides listed in Supplementary Table S1. Amplification of the 5’ untranslated region (UTR) with the primer combination 5’-hmgb2-for and 5’-hmgb2-rev including ApaI and SmaI restriction sites, respectively resulted in a 526-bp fragment and was cloned upstream to the positive selection marker human dihydrofolate reductase (hudhfr) previously introduced into the pBC SK- vector (Stratagene) under the control of EF1α promoter and of dhrf/ts 3’UTR (dihydrofolate reductase/thymidylate synthase). Next, the 3’UTR region was amplified with 3’-hmgb2-for and 3’-hmgb2-rev primers including NotI and AscI restriction sites. This fragment was inserted downstream to the hudhrf box resulting in the hmgb2 targeting vector pBC-5’B2hudhfr3’B2 allowing replacement of the endogenous hmgb2 locus in PbANKA upon a double cross-over homologous recombination and subsequent selection with the antifolate pyrimethamine. P. berghei parasite transfections were performed with 5 μg ApaI/AscI-digested pBC-5’B2hudhfr3’B2. |
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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