siRNA templates from the Human 8.5K siRNA Library in pFIV-H1-copGFP were packaged in viral particles, amplified, labeled, and hybridized to the Affymetrix Genome Focus Array. Similarly, siRNA templates were amplified from the genomic DNA extracted from target cells transduced with the packaged library.  The data demonstrates that the transduction of the library is highly efficient and does not bias the library with respect to representation.

GeneNet^TM siRNA Libraries consist of many thousands of siRNA templates cloned into packaged pFIV™ Lentiviral Vector constructs. There are several siRNA sequences targeting each gene. Thus, the GeneNet 1.5K Human Cancer Library was constructed with 7,500 different siRNA sequences, and the 8.5K libraries were constructed with more than 40,000 different siRNA sequences.

In addition to optimizing the siRNA template design for things like favorable GC content and no termination signals, all current GeneNet™ Libraries contain siRNA sequences that hybridize to probes present on Affymetrix GeneChip® Arrays. This design facilitates detection of the sequences after stable integration into the target cell genomes.

GeneNet libraries are constructed using standard cloning techniques. Briefly, the siRNA templates are synthesized with the ends that are ligation-compatible with the digested pFIV library vector. Specific details of the restriction enzymes, insertion sites, and the oligonucleotide designs vary from library to library and are provided with the documentation for the specific libraries.

Following construction, each library is packaged into highly transducible VSV-G pseudotyped lentiviral particles by passage through packaging cells that are co-transfected with the pFIV-PACK™ Lentiviral Packaging Kit. All GeneNet Libraries are provided in a pre-packaged, transduction-ready, ready-to-use form.

After construction, representation of the libraries is confirmed by amplifying, labeling, and hybridizing to Affymetrix arrays the siRNA templates isolated from cells transduced with the library. These hybridization results are also compared with similarly prepared labeled and hybridized siRNA templates from the packaged viral supernatant used to transduce the target cells. An example of this data is shown. Specific QC data for each library is provided with the library documentation. This data confirms the presence of a majority of the siRNA templates used in constructing the library. However, many of the siRNA templates not seen in this analysis may still be present in the library. They simply may not amplify, label, or hybridize well enough to be detectable in the complex population. Some of these siRNA may be detected in a smaller population derived from cells selected for a desired phenotype.