Figure 5 Northern blots of small RNAs extracted from Igl and PATMK transfectants. To test if the U6 promoter was driving hairpin expression, shRNA transfectants (PATMK (3552–3580), PATMK (2273–2301), SHP099 mouse PATMK (3552–3580 scrambled) [39], Igl (1198–1226), Igl (2412–2440), and Igl (2777–2805) were selected with 30 μg/ml hygromycin for 48 hours before check details harvesting. HM1:IMSS non-transfected amebae were included as negative controls. Small RNAs were extracted using the mirVana™

miRNA Isolation Kit (Ambion) (Applied Biosystems/Ambion, Austin, TX, USA). Fifty μg small RNA were loaded per lane on a 12% denaturing acrylamide gel and transferred to membrane. rRNA bands were analyzed to ensure equal RNA

loading. Oligo probes matching to the sense and antisense strands of the hairpins were end-labeled with 32P and were hybridized with each corresponding sample blot overnight at 37°C overnight, washed with low and medium stringency conditions, and exposed overnight to film. Note the two product sizes, which Proteases inhibitor may correspond to the unprocessed hairpin (~60–70 nucleotides) (blue arrows) and the processed siRNA products (~30 nucleotides) (red arrows). Discussion We have utilized the U6 promoter to drive expression of shRNAs with a 29-bp stem and a 9-nt loop to knock down protein expression of three unrelated genes: a membrane protein, Igl, the intermediate subunit of the Gal/GalNAc lectin; URE3-BP, a calcium-regulated transcription factor, BCKDHA upstream regulatory element 3- binding protein; and EhC2A, a membrane-binding protein. Previously we had reported preliminary experience with this system in the near-complete knockdown of phagosome-associated transmembrane kinase 96 (PATMK) [39]. In the work reported here, the highest level of protein knockdown for Igl was 72%, for URE3-BP 89%, and for EhC2A 97%. We concluded that this was a reliable and effective system for gene

knockdown in E. histolytica. This method has advantages over other methods used for gene silencing: the U6-shRNA expression cassettes are small (420 bp), appear to be active against different types of genes, yield significant knockdown, and the expression vector, once transfected, allows continuous expression of shRNAs, thus avoiding performing multiple transfections, and the shRNAs can be easily synthesized via PCR. Not every transfected shRNA construct was equally effective in silencing gene expression. For example, neither the EhC2A (502–530) nor the Igl (2412–2440) shRNA construct blocked gene expression. In the case of Igl (2412–2440), the run of four thymidines at positions 19–23 in the shRNA sense strand could possibly cause RNA polymerase III to terminate the transcript prematurely.