Custom Pooled Lentiviral Libraries
Oligonucleotide and gene synthesis technology have largely made more traditional techniques to clone genomic DNA and RNA isolated directly from organisms, obsolete. Traditional cDNA, genomic, and other fragment libraries have become antiquated—-replaced by well-defined arrayed or pooled libraries made from synthesized sequences. In addition to improving the efficiency and quality of cloned constructs and libraries, these synthetic sources of genetic sequences have enabled researchers to design and build defined libraries of elements that are not naturally occurring, such as barcoded sequences, novel promoters, and CRISPR sgRNA.
As one of the pioneers in creating libraries with synthetic oligonucleotides, Cellecta has developed a proficiency in producing high-quality libraries expressing short genetic elements, such as shRNA, peptides, enzyme active site elements, and sgRNA. While Cellecta uses standard proven cloning techniques to generate these libraries, there are many technical challenges that need to be overcome to fully produce libraries with representative, evenly distributed elements that are amenable to next-generation sequencing read-outs.
Cellecta primarily focuses on:
- pooled shRNA libraries for RNA interference (RNAi) screens
- heterogenous barcode libraries for cell labeling
- CRISPR knockout, activation (CRISPRa), and inhibition (CRISPRi) sgRNA libraries for genetic disruption and activation screens
We have also developed:
– peptide expression libraries
– miRNA libraries
– promoter libraries
– cDNA libraries with active domain variations
Using the same basic approach described below, we can provide virtually any type of pooled library where the variable element can be defined within a couple-of-hundred-base sequence. Combining this with sophisticated cloning approaches enables us to produce a large variety of libraries, with barcodes if desired, in almost any vector.
Contact us to explore your options for developing a pooled library for any sort of screen.
Library Construction Process:
- Design and synthesize oligonucleotides for cloning. Oligonucleotides may be designed to encode shRNA or sgRNA (as shown in the graphic), a peptide sequence, a portion of a larger protein, or may correspond to promoter variations, barcodes, or other genomic elements.
- Library cloning into the vector of choice. Once synthesized, oligonucleotides are cloned into the appropriate vector for the biological model and planned screening study. Cellecta has a selection of standard lentiviral vectors with different markers for typical libraries. However, we can make libraries in customer provide vectors, as well.
- Confirmation of library quality. After cloning, the distribution of cloned oligos in the pooled library is assessed using next-generation sequencing (NGS).
- Packaging for libraries made in lentiviral vectors. As an optional service, Cellecta can also package your library as VSV-g pseudotyped lentiviral particles. Various packaging scales are available.
Email us for more information.