The control GFP sequence [30] was used to design oligos for making a shRNA MK-2206 in vivo control construct. Sense strand sequences chosen to make the Igl, URE3-BP and EhC2A shRNA constructs

successfully transfected into trophozoites are shown in Table 1, and PCR oligos used to amplify these sequences to generate shRNAs via PCR are shown in Table 2. PCR conditions for generating shRNAs Initially, E. histolytica genomic DNA was used as a template for the first round of Igl shRNA PCRs. For the URE3-BP and EhC2A shRNA PCRs, the cloned U6 promoter was used as the PCR template: the Igl shRNA plasmids were digested with HindIII and ApaI and the U6 promoter was gel-purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). Two rounds of PCR were used to generate the shRNA constructs. The first PCR round BAY 11-7082 order generated the sense strand of the hairpin and the loop. Reaction volumes of 40 μl were set up, each consisting of 0.6 μl SAHARA™ DNA polymerase (Bioline USA Inc., Taunton, MA, USA), 4 μl 10× SAHARA™ PCR buffer, 3.2 μl 50 mM MgCl2, 2 μl dNTP mix (stock 10 mM each), 0.4 μl U6 HindIII forward oligo (100 μM stock), 0.4 μl R1 oligo (100 μM stock), 1 μl (200 ng E. histolytica genomic DNA or 25 ng gel-purified digest Combretastatin A4 nmr of HindIII/ApaI U6 promoter), and 28.4 μl sterile water. Cycling conditions were as follows: 95°C for 8 minutes, 10 cycles of 95°C 45 sec, 40°C 1 min, 68°C 1 min 30 sec;

25 cycles of 95°C 45 seconds, 52°C 1 min, 68°C 1 min 30 sec, and a 5 min final extension Mirabegron at 68°C. 5 μl of each PCR product was subjected to agarose gel electrophoresis to check that the products were ~380 bp. In the second PCR round, the first round PCR product was used as a template to add the antisense strand of the hairpin, the terminator sequence and the NotI site. Each 100 μl-volume reaction contained 2 μl SAHARA™ DNA Polymerase (Bioline USA Inc., Taunton, MA, USA), 10 μl 10× SAHARA™ PCR buffer, 8 μl 50 mM MgCl2, 5 μl dNTP mix (10 mM each), 0.8 μl U6 HindIII forward oligo (100 μM), 0.8 μl R2 oligo (100 μM), 2 μl PCR product from the first PCR round, and 71.4 μl sterile water. Cycling conditions were:

95°C for 8 minutes, 10 cycles of 95°C 45 sec, 18.5°C 1 min 30 sec, 68°C 1 min 30 sec; 30 cycles of 95°C 45 seconds, 55°C 1 min, 68°C 1 min 30 sec, and a 5 min final extension at 68°C. The low annealing temperature in the early cycles of the second PCR was used since the loop is the only overlap between the first round product and the second round reverse oligo. The second round PCR products were checked by agarose gel electrophoresis for products of the correct size (~420 bp). Sometimes a smaller product was present in addition to the correct size product in the final PCR product; this was ignored since it had no effect on the subsequent cloning steps. These final PCR products were ethanol-precipitated, then they and modified pGIR310 were digested with HindIII and NotI.