Human Whole-Genome ORF Expression Library
Empowered by our human whole-genome Open Reading Frame (ORF) collection, VectorBuilder offers ORF library construction and screening services for various overexpression screening needs. With each individual ORF clone fully sequence validated, we can rapidly construct custom screening libraries in any backbone, in pooled or arrayed formats, and experiment-ready as plasmid or viral particles in a cost-effective way. Additionally, each ORF in the library can be uniquely barcoded to facilitate screening. Ideal for gain-of-function studies and validating CRISPR or RNAi results, our ORF libraries are valuable tools for investigating gene function, disease mechanisms, and therapeutic responses, as well as streamlining the drug discovery process.
Highlights
Genome-wide collection of >16,000 sequence-validated human protein-coding ORFs makes the ORF expression libraries highly affordable with fast turnaround.
Available in pooled and arrayed formats and can be delivered as E. coli glycerol stock or ready-for-screening plasmid or high-titer virus.
Powerful high-throughput pooled library screening available in vitro and in multiple in vivo models including mice, rats, and non-human primates (NHPs).
Comprehensive tech support on library design and screening strategy
for unbiased and scalable screening, multiplexing analysis, and more.
Talk to our experts to design your ORF library now!
Selecting genes from collection
Library cloning
Library QC
Virus
packaging
packaging
In vitro or in
vivo screening
vivo screening
Hit identification
Service Details
| Service Module | Description | Price | TAT |
|---|---|---|---|
| Library Design | Our process starts with a consultation where our experts will help design the right library for your needs. We guide you to select the right delivery system, choose the appropriate promoter, tag, and marker depending on your screening strategy, and optimize vector design for achieving high signal-to-noise ratio and unbiased readout. | Free | 1-4 days |
| Library Cloning | We conduct massive parallel cloning of sequence-verified ORFs into the desired vector backbone followed by stringent QC. For pooled libraries, we guarantee high fold coverage and uniformity. Deliverables include E. coli glycerol stock or plasmid or high-titer virus. Arrayed libraries can be delivered in 96- or 384-well plates or matrix tubes. | Please inquire | 2-3 weeks |
| Virus Packaging | We package your library plasmids into high-titer viruses with high-fold and uniform coverage. Find detailed information about our virus packaging services here. | Please inquire | |
| Pooled Library Screening | We can conduct streamlined in vitro or in vivo screening of your pooled ORF libraries. The in vivo screening can be performed in rodent models or non-human primate (NHP) models. We can insert barcodes to facilitate the readout at DNA or RNA level. | ||
| Hit Identification | We take care of post-screening steps with preparation of genomic DNA or RNA from screened cells, NGS sequencing, and hit analysis. We deliver raw and processed sequencing data as well as project reports to you. | ||
Technical Information
Gene list
Our ORF collection includes >16,000 genes with lengths ranging from 100 bp to 10000 bp, covering multiple categories and canonical pathways such as cell growth and proliferation, immune response, development and differentiation, and more. You can download the complete list of genes included in our libraries here.
Library design
A delicate library design is the foundation of a successful screening experiment. Some key considerations while designing your ORF library include:
- Gene delivery system: Viral and non-viral delivery systems for ORF libraries provide distinct advantages and limitations. Of the non-viral systems, regular plasmids are simple and cost-effective, ideal for transient expression. PiggyBac is a non-viral, transposon based system that integrates into the genome and provides a high transgene carrying capacity. Of the viral systems, lentiviral vectors allow for stable integration into the genome, enabling long-term expression. AAV is a non-integrating viral delivery system that offers high safety and efficiency, particularly in vivo, but with a limited packaging capacity.
- Promoter: Depending on your experiment, you might need a constitutive, inducible, or tissue-specific promoter. For instance, in vivo screens often benefit from use of a tissue-specific promoter. Read more about promoters here.
- Drug-selection marker: Including antibiotic resistance genes in the vector is necessary for selecting successfully transformed cells. If you work with cell lines that already have antibiotic resistance (e.g. 293T cells are neomycin resistant), make sure you pick a different selection marker for your library. We generally recommend the puromycin-resistance gene for its fast and effective selection. Information about other options can be found here.
- Protein tag: Adding tags like His- or FLAG-tags or fusion proteins like GFP or mCherry can help with the detection, purification, and localization of the expressed ORFs. VectorBuilder’s collection of tags can be browsed here.
- Fluorescent/luminescence reporter: Adding fluorescent or luminescent reporters to the ORF can help with visualization or cell sorting, enabling tracking of cellular events in real time. Depending on experimental needs, these reporters can either be fused directly with an ORF for localized tracking of the target protein or expressed under a different promoter for non-localized, independent monitoring. Learn more about reporters here.
- Barcode: Using cell and molecular barcodes allows for cost-effective analysis of thousands of ORFs in a pooled screening experiment. Designing the placement of a barcode in a vector involves strategic considerations to ensure effective readout and compatibility with library cloning, screening, and deconvolution processes.
Our design team can work with you to customize the ORF library for your specific needs. Send a design request to learn more.
Pooled vs. arrayed libraries
VectorBuilder ORF libraries can be delivered in a pooled or arrayed format, both involving different approaches and advantages.
| Attribute | Pooled Library | Arrayed Library |
|---|---|---|
| Construction | All barcoded ORF-carrying plasmids/viruses pooled into a single mixture | Individually isolated ORF-carrying plasmids/viruses placed in multi-well plates |
| Screening | High-throughput screening | Systematic study of each ORF |
| Approach | Across thousands of ORFs simultaneously, with reference and experimental groups | Independently, one ORF per well |
| Advantages | Quick, cost-effective for large-scale studies, allows simultaneous analysis of numerous ORFs | Precise control over experimental conditions, reduces cross-contamination |
| Limitations | Challenging to identify specific ORFs responsible for phenotypes without additional deconvolution and validation steps | More labor-intensive and expensive due to individual handling and analysis |
In vitro and in vivo screening
Pooled screening of the ORF library for identification of enriched genes can be conducted by the expert team at VectorBuilder in both in vitro and in vivo systems. In vitro screening is rapid and technically straightforward, and conducted using your chosen cell lines that allow for quick identification of genes with differential expression. A potential limitation in translatability of vectors between in vitro and in vivo applications exists as vectors optimized for in vitro conditions may not exhibit the same efficiency in vivo, and those showing specific expression patterns in vitro might behave differently in vivo.
In vivo animal models can offer a more reliable platform for screening ORF libraries in cell types that are difficult to culture or exist within complex tissue structures. While both mice and NHPs are used for in vivo screening, NHP models are preferred due to their high degree of similarity to humans, which can help ensure higher efficacy of gene therapy applications. Various administration routes are available for in vivo screens, including tail vein, facial vein (for neonatal mice and rats), intracerebroventricular, and subretinal, intravitreal, intratympanic, and intramuscular injections, among others. All of our animal experiments are conducted in AAALAC-accredited facilities.
After delivering the ORF library to target cells or animals, the activity of candidates is assessed by measuring the expression of a reporter gene or barcode. Targets can undergo multiple rounds of screening to ensure robust identification. Following screening, deconvolution is performed by isolation of genomic DNA or total RNA followed by sequencing and enrichment/depletion analysis.
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