We routinely select specific RNAi gene targets (400–600 bp) and primers using the RNAit software http://trypanofan.path.cam.ac.uk/software/RNAit.html. A single pair of PCR primers are designed that incorporate four selected restriction sites (not present in the RNAi target fragment) such that a single PCR product can be differentially digested and sequentially cloned. For example, using MCS1/2, the following primers could be used to clone antisense followed by sense fragments: Primer 1, XbaI–BamHI-5′ sequence; Primer 2, ApaI–KpnI-3′ sequence. The antisense and sense fragments are generated by BamHI–KpnI and XbaI–ApaI digestion respectively. Selectable marker (HYG) transcription is then constitutive (PEP: procyclin promoter) and independent of the Tet-regulated cassette (PRRNA: RRNA promoter). We maintain 2T1 T. brucei cells (Lister 427, MITat1.2, clone 221a), that also express the Tn10 Tet repressor (TetR::BLE::TUB), in HMI-11 growth medium containing phleomycin and puromycin (0.5 µg ml−1 and 0.2 µg ml−1 respectively). We use electroporation (Amaxa) with ∼2.5 × 107 cells and ∼3 µg of each AscI-linearised construct in duplicate; each mixture is transferred to 36 ml of medium in a 25 cm2 culture flask. After ∼6 h, the appropriate drug selection is applied (2.5 µg ml−1 hygromycin) and cells are distributed into 12- or 24-well plates. Each electroporation experiment typically, and conveniently, yields 2–5 transformed clones using this approach and these are visible after ∼5 days (∼100,000 cells). To screen clones for simple, double-crossover integration and loss of the PAC gene, we remove ∼5% of each day-5, hygR culture and grow with or without puromycin (2 µg ml−1 ) in a 24-well plate for another 24–48 h. Typically, ∼50% of clones are puromycin sensitive and two of these are retained for further analysis. Note that these strains are now resistant to phleomycin (TetR::BLE::TUB) and hygromycin (HYG::RPa::RRNA).
Filename: Generation of RNAi or Overexpression cell lines in T.brucei 2T1 cell line.doc
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Created: 25th Feb 2012 at 13:35
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