Pooled lentiviral library content
* 150,000 siRNA sequences targeting 39,000 mouse mRNA transcripts
* Selectable puromycin resistance allows positive selection of transduced cells
* Pre-packaged, ready-to-use format—transduce your cells the day you receive the library
* High-throughput identification of functional siRNAs by Affymetrix GeneChip® hybridization
* Choose from siRNA libraries in FIV or HIV-based lentivectors
* Also available in plasmid form, you can now produce large quantities of packaged siRNA library in your own lab
The GeneNet™ Mouse 40K siRNA Library contains 150,000 siRNA templates targeted to 39,000 mouse transcripts listed in the NCBI RefSeq database. For most of the target genes, four different siRNA sequences were designed using our proprietary algorithm. The library is available cloned into the FIV-based pSIF1-H1-Puro or HIV-based pSIH1-H1-Puro shRNA Expression Vectors, which confer resistance to the antibiotic puromycin. This expresses cloned short-hairpin RNA (shRNA) from a single H1 RNA polymerase III promoter.
The library is available in both pre-packaged and plasmid form. The pre-packaged VSV-G pseudotyped viral stock can be used to transduce your cells of choice immediately upon receipt, or be stored at -70oC for several months. With the library in plasmid form, you can perform the pseudoviral packaging step in your own cell culture facility. Enough plasmid is provided to yield about 109 ifu of packaged siRNA library.
After transducing the library into your cells of interest, you select those cells that display the desired phenotype or response using whichever technique is appropriate for your system. Following selection, the lentiviral inserts—containing the siRNA templates—are easily recovered by PCR and identified using the GeneChip® Mouse Genome 430 2.0 Array (Cat.# 900495) manufactured by Affymetrix (Santa Clara, CA). The siRNA template sequences were designed to hybridize to oligonucleotide probes on this array to facilitate identification. Although more tedious, you can also use sequencing to identify the particular siRNA sequences in selected cells.
To confirm representation, siRNA templates for the library were packaged in viral particles, amplified, labeled, and hybridized to the Affymetrix Mouse Genome 430 2.0 Array . Similarly, siRNA templates were amplified from the genomic DNA extracted from target cells transduced with the library. These templates were also labeled and hybridized to the Mouse Genome 430 2.0 Array. Overall, in both the viral- and cell-derived samples, more than 55% of the gene-specific siRNA template species used in construction of the library were easily detectable above background. In addition, the correlation of the relative amounts of different siRNA template species between both samples was very high, indicating that there is not appreciable loss or change in representation with transduction. These results indicate that the library delivers a representative number of siRNA sequences that target the majority of genes for which the library was originally designed. Also, the high correlation between the pre- and post-transduction samples shows that the pseudotyped viral particles transduce with similar efficiency, regardless of the siRNA template they harbor.