Gene Modulation

Contact: Phil Corrin
Location: Eastlake Building E1-310
Contact phone: (206) 667-4670
Contact fax: (206) 667-2825
Contact e-mail: pcorrin@fredhutch.org

Gene modulation technologies include RNA interference, DNA editing and expression, and chemical compounds that suppress, enhance, or modify gene expression. These can be in the form of RNA, DNA, or protein, and can be introduced into cells in culture through direct application to media, lipofection, electroporation, or viral transduction.

The Genomics Shared Resource Gene Modulation Lab provides material support, consultation, and training in the use of gene modulation technology and precision, high-throughput liquid-handling systems with applications ranging from high-throughput screens to investigating individual gene function and custom protocol development.

Investigators interested in adding this technology to their existing experimental systems should contact the Genomics Shared Resource to set up an introductory consultation. For questions please contact Jeff Delrow (jdelrow@fredhutch.org or (206) 667-2763) or Phil Corrin (pcorrin@fredhutch.org or (206) 667-4670).

Gene Modulation Technology

The Genomics Shared Resource supports the use of various platforms to manipulate gene expression:

Services include provisioning materials, hairpin and sgRNA design, development of cloning methodology, custom vector design and experimental modeling, and consultation and training in each of these areas. We are continuously working with labs to evaluate and develop new platforms to expand the experimental reach of gene modulation systems, and can assist experienced users of these tools as well as provide introductory training to labs wishing to acquire them.

Gene knockdown

RNA interference (RNAi) involves the use of small interfering RNA (siRNA) or short hairpin RNA (shRNA) to target mRNA and reduce gene expression. We have siRNA and shRNA libraries for high-throughput genome wide screens, individual hairpins in GIPZ and other vectors to validate screen hits and investigate gene function, and provide training in the use of this material. We also support custom hairpin design to expand the shRNA target list beyond what is available in the library. Custom derivatives include multiplexed hairpins to target multiple genes with a single construct or to modulate the level of knockdown for a single gene, and marker/selector panels to provide experimental flexibility. An online searchable database and ordering system has been established to facilitate purchase of individual clones. Please note that access to the collection is limited to Fred Hutch labs only.

Gene knockout

CRISPR technology can be used to directly target DNA and cause mutations that result in premature stop codons which can knock out gene expression. We have control sgRNAs cloned into a panel of CRISPR/Cas9 backbones so labs can evaluate the platform for use in their existing experimental models. Custom derivatives include CRISPR/Cas9 in the PiggyBac transposon system and deactivated Cas9 fused to fluorescent proteins or functional enzymes for delivery to a specific genomic address. We support the entire design and synthesis pipeline from target evaluation to single or bulk vector assembly methods, and provide training in the production and use of this material.

Gene expression

Gene expression systems can be transient or integrate into the genome, and range from CRISPR/Cas9 based synergistic activators to commercially available ORF expression vectors. Custom ORFs can be PCR amplified from commercial vectors or cDNA, or synthesized as double stranded DNA (i.e., gBlocks) for cloning into expression vector backbones. Custom derivatives include knockdown/knockout resistant ORF variants for use in mutant allele rescue experiments, or ORFs fused to fluorescent tags for gene product tracking and localization assays. We support a variety of assembly and expression methods, and provide training these areas.

Gene tagging and targeting

There are many CRISPR/Cas9 mediated targeting schemes available and it is important to determine which will work best in a particular model system. Manipulations range from altering a single base or adding a short tag, to targeting an expression cassette to a precise genomic location. We support a range of design, synthesis and application methods involved with this process, and provide training in these areas.

High-Throughput Screening

The Genomics Shared Resource operates a state-of-the-art High-Throughput Screening laboratory (HTS). The HTS lab conducts well-by-well screens using commercial libraries and custom collections:

Screens are supported by specialized high-throughput liquid-handling robotics and detection systems, including:

Other detection systems available to Fred Hutch's HTS lab include:

Custom Protocol Development

Robotic liquid handling allows for bulk processing of replicates as well as precise, repetitive manipulation of materials. This is particularly useful for technique sensitive operations like magnetic bead purification of a large number of samples, or working with reagents that have a limited functional lifespan between the initial mixing of components and final application to target cells in a large number of plates.

Cost benefits can be achieved by scaling down reaction sizes and robotic manipulation can reduce variability in process output. The Genomics Shared Resource works with labs to translate their existing protocols into high throughput procedures and to develop novel processing methodology.

Processes currently available:

  • Plate based sample array, re-array, and dilution
  • Magnetic bead sample purification
  • High throughput cell plating (96 or 384 well plates)

Processes currently under development:

  • AutoCUT&RUN (in conjunction with Henikoff lab)
  • CRISPR/Cas9 mediated chromatin isolation, purification, and sequencing
  • High-Throughput cloning

Doing Business with Genomics

Thomas Bldg DE-740
(206) 667-2714
genomics@fredhutch.org

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