Figure 3

Generation of TbAGO1-/- (KO) and TbAGO1-/- +GFP::TbAGO1 ^Ticell lines. A. Schematic representation of the endogenous TbAGO1 locus (top), and of the constructs used for double TbAGO1 knock-out (middle) and for expression of GFP::TbAGO1 fusion protein (bottom). Large and small boxes represent protein coding sequences and TbAGO1 flanking regions used for the knockout construct. The tetracycline-inducible EP promoter of the pGFP::TbAGO1430 vector is indicated by a star. B. Southern blot analysis showing the absence of TbAGO1 from the TbAGO1-/- (KO) cell line. Genomic DNA was purified from the indicated cell lines, digested with Sca I and Xba I, and fragments were separated and transferred on a nylon membrane for hybridisation with a TbAGO1 probe. C. Northern blot analysis. Total RNA (10 μg) from the indicated cell lines was run on a gel and transferred onto a nylon membrane for hybridisation with a TbAGO1 probe (top panel) or with an α-tubulin probe (central panel). The bottom panel shows ethidium bromide staining of the membrane after transfer.