shRNA system for Entamoeba histolytica. (A) Diagram of the two-step PCR process for generating short hairpins shRNA constructs were made using the method of Gou et al (2003) . Genomic DNA (or subsequently, the cloned U6 promoter) was used as a template to amplify the E. histolytica U6 promoter and to add the hairpins. The primers in the first PCR round were the forward primer, containing a HindIII site and 5' end of the U6 promoter, and a first reverse primer, containing the U6 promoter 3' end, the shRNA sense strand sequence, and the 9-nucleotide loop. To yield the final product, in the second PCR round, the same forward primer was used, with a second reverse primer containing the loop sequence, the antisense strand sequence, the termination sequence, and a NotI recognition site, using the first round product as a template. The primers used to generate the PCR products are listed in Table 2. (B) Modification of amebic expression vector pGIR310 to express shRNA The tetracycline repressor cassette in expression vector pGIR310, a modification of pGIR308 [49, 50], was replaced with a polylinker containing a SalI and NotI site, flanked by HindIII sites. PCR products were cloned into the HindIII and NotI sites. pGIR310 confers hygromycin resistance in amebae and ampicillin resistance in E. coli bacteria. (C) Expected structure of 29-basepair shRNA before processing by Dicer The 29-basepair stem and 9-nucleotide loop are shown.