Pooled shRNA Library Construction
Cellecta's partnership with Agilent Technologies has allowed us early access to the Oligonucleotide Library Synthesis ("OLS") technology program which has been vital in the development of our pooled shRNA libraries. We tested the oligo libraries of several HT oligo synthesis service providers, and the results showed a dramatic difference in quality. The mutation rate during synthesis of Agilent's oligos was an order of magnitude less than that of any other provider. As a result, we now have arguably the best quality pooled shRNA libraries available on the market.
Optimized Vector and shRNA Design
The shRNA design used in the making of our validated shRNA libraries involved the use of proprietary software for in silico prediction of functional shRNAs, a database of validated shRNA, and optimized designs of both expression vectors and shRNA. The vector and shRNA design alone, used in our custom shRNA libraries, delivers approximately 55-70% functional shRNA (>70% knockdown) depending on the cell type. In comparison, the percentages of functional shRNA for two other major shRNA library providers are 50% (21-mer shRNA design) and 20% (miR30 design).
To provide efficient delivery of complex shRNA libraries into different cell types, we have developed third generation HIV-based lentiviral shRNA cloning vectors with H1 or U6 tet-regulated or constitutive promoters for expression of shRNA and a choice of a single or dual selection marker (GFP, RFP, PuroR, BleoR, etc.) expressed from a single CMV, EF1, PGK, UbiC, or other promoter (Figure 1).
Figure 1. Diagram of lentiviral shRNA library vector indicating customizable options.
Any Set of Genes, at Any Redundancy
We will generate lentiviral pooled shRNA libraries, for any set of genes, using an optimized shRNA design and unique shRNA-specific bar-codes. A redundancy of approximately 5-10 shRNA per mRNA is recommended, but not required. We can design any number of shRNA per gene, limited only by the size of the mRNA target transcript. Alternatively, choose one of our pre-designed sets of shRNA targeting the human or mouse druggable genome, critical signaling pathway genes, or cancer-specific genes. All libraries are designed to contain sequencing primer binding sites (Gex1, Gex2) and shRNA-specific, 18-nt bar-codes compatible with the Illumina HT Sequencing platform (Figure 2).
Figure 2. Diagram of shRNA cassette. Any set of shRNA can be designed, and unique shRNA-specific bar-codes will be used for detection by Illumina HT Sequencing. Triplicate bar-coding is recommended to improve statistical significance of data.
Cellecta's HIV-based lentivectors are similar in design but have improved knockdown efficiencies in comparison with the other HIV-based vectors successfully used by others for cloning and expression of shRNA and shRNA with microRNA30-like features. Our lentivector system allows for the production of pseudoviral particles with similar titers (1 – 10 × 106 ifu/ml) during the packaging step, but these lentiviral particles have different tropisms and are varied in the efficiency of transduction in a wide range of cell lines. Figure 3 outlines the technology developed during preliminary studies for construction and quality control of genome-wide lentiviral shRNA libraries.
Figure 3. Construction and Q.C. of pooled lentiviral shRNA libraries. Cellecta's bar-coded shRNA libraries make use of Illumina HT sequencing as the detection method, unlike other systems which use a microarray hybridization step or individual sequencing of selected clones. Digital HT sequencing readouts eliminate the problem with low dynamic range of microarray data, reducing the number of false positives and negatives and increasing the number of real positive hits.
Stringent Quality Control Criteria
Cellecta's custom shRNA library Q.C. procedure consists of insert screening, mutation rate determination, and an optional verification of representation by high-throughput (HT) sequencing. The quality control criteria include:
- High insert rate: At least 90% of clones have inserts
- Very low mutation rate: less than 0.2% in shRNA sense (active) portion, i.e. 2 mutations/deletions per 1,000 nucleotides, or less than 10% of constructs have mutation/deletion in sense portion of shRNA
- Excellent library representation: Typically, 90% of shRNA constructs in the library are represented within a 100-fold abundance range. Cellecta guarantees replacement if it is less than 80%.
View our Frequently Asked Questions (FAQs) page for detailed information on our pooled lentiviral shRNA libraries. For relevant references and literature, visit the Articles page under Resources.
Please read the Cellecta-Agilent License Statement for the terms of use.
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