Clone size of randomized shRNA-libraries.

<p>To indicate the complexity of the shRNA library, the number of bacterial colonies of each transformation step (primary library, secondary library and final library) was documented as colony forming units (cfu).</p

Specificity of shRNAs against HIV-1.

<p><b>a)</b> Designation of the identified shRNAs <b>b)</b> shRNA-target sequence of the HIV-1 proviral clone pNL4.3 <b>c)</b> starting nucleotide of the shRNA based on +1 being the transcriptional start site. <b>d)</b> Number of known isolates which include the target sequence.</p

Construction of the shRNA-library.

<p><b>a</b>) The 3 HIV segments of pNL4.3 were fragmented using DNaseI and blunt-ended. <b>b</b>) Fragments of 150–300 bp were eluted and ligated to the 3′ loop. To limit the size of the HIV-1 inverted repeats, a recognition site for MmeI, which cleaves exactly 20nt from its recognition site and leaves 2 nt 5′ overhangs, was engineered into the 3′ loop. <b>c</b>) Ligation of the 5′ loop to the MmeI-digested fragments generated a quasi-circular single-stranded structure. <b>d</b>) Rolling circle amplification (RCA) reactions using Φ 29 DNA polymerase and the primers RCA1 and RCA2 were performed to amplify the single-stranded circular DNA and to generate the complementary strand yielding a DNA concatemere of palindromic, inverted repeats encoding siRNA molecules. Digestion with BglII and MlyI liberated shRNA sequences which were inserted into the expression vector pENTR/siLib. <b>d</b>) The shRNA sequences were cloned into the linearized pENTR/Lib generating the primary library which was religated after BamHI digestion yielding the secondary library. The final lentiviral shRNA-library was generated by LR recombination of the secondary library into pL/EGP/siLib.</p

Reconfirmation of the inhibitory potential of selected shRNA-sequences.

<p><b>a</b>) ShRNAs were PCR-amplified from genomic DNA of selected cell clones as shRNA expression cassettes consisting of H1 promoter, shRNA-sequence and polyT. Each of the 200 individual cassettes were co-transfected with the HIV-1 specific Luciferase reporter construct pNL4.3Luc.R-E- into HEK 293 FT cells. Luciferase expression was measured 48 h p.t. Cells transfected with pNL4.3Luc.R-E- and a scrambled shRNA (sh scr) expressing cassette were used as control. <b>b</b>) Map of the identified shRNAs and re-evaluation of their inhibitory potential upon co-transfection as described in <b>a</b>). Cells which were transfected with pNL4.3LucR-E- alone or co-transfected with a scrambled siRNA control served as controls. Error bars indicate +/− SD of mean of three independent experiments.</p

Expression of the shRNAs from different polymerase III promoters.

<p><b>a</b>) 6 potent shRNA sequences were subcloned to allow for U6 promoter driven shRNA expression. U6 or H1 promoter expression vectors and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells. Western blot analysis with a HIV-1 Integrase specific antibody 48 h p.t. indicated that the shRNAs can be efficiently expressed from both polymerase III promoters. Actin was used as loading control. <b>b</b>) <b>c</b>) Published shRNA-sequences against pol, nef, rev/env, gag and vpu/env were cloned to be expressed by the U6 promoter. These constructs as well as 14 library shRNAs and pNL4.3LucR-E- were co-transfected into HEK 293 FT cells and analysed 48 h post transfection. Western blot analysis <b>b</b>) with a HIV-1 Integrase specific antibody or <b>c</b>) luciferase assays demonstrated that the newly identified shRNAs are as potent as the published shRNAs. A scrambled shRNA (sh scr) and non-transfected cells were used as controls.</p