Note, the core is closed.
Renate Lewis, director of the Transgenic Vectors Core, is retiring and the core will close. New projects can be accepted until Friday February 7, 2020.
The Transgenic Vectors Core has worked in close collaboration over the years with the Mouse Genetics Core (Mia Wallace). The Mouse Genetics Core will continue to provide CRISPR and TALENS gene editing injection services. Please contact Mia Wallace for questions about new projects. (Injection services also are provided by the Injection Lab in the Department of Pathology & Immunology.)
Renate, many thanks for your years of service, and your tireless efforts to make available cutting-edge resources in vector construction!
Mouse transgenesis, the stable integration of foreign DNA into the mouse genome, is a key approach used in biomedical research to explore gene function and regulation and to model human disease. Innovations in mouse transgenesis technologies include conditional gene modifications that inactivate gene function in tissue-specific manners, thereby overcoming embryonic lethality; and BAC transgenes to circumvent the position effects commonly found in standard transgenic mice. More recently, TALENs (TAL effector nucleases) and CRISPR/Cas9 systems have emerged as powerful new tools for genome modification in a variety of organisms and cell types, including mouse, rat, zebrafish, drosophila, C. elegans, and human stem cells and iPSCs (induced pluripotent stem cells).
The Transgenic Vectors Core specializes in novel genome engineering technologies and vector construction using recombineering and isothermal assembly to generate complex gene modifications not possible with restriction enzymes. Large segments of genomic DNA such as those found on BACs (Bacterial Artificial Chromosomes) are now routinely modified, facilitating production of vectors that were previously impossible to generate.
The Transgenic Vectors Core designs and constructs TALENs, CRISPR/Cas9 and gene targeting vectors on a fee-for-service basis. Services are available for use by all Washington University investigators as well as investigators outside of the Washington University community.
TALENs and CRISPR/Cas9 vectors
TALENS (TAL effector nucleases) and CRISPR/Cas9 systems are engineered nucleases that promote double stranded breaks in the genome, stimulating gene targeting by activating cellular repair pathways. With targeting efficiencies as high as 75%, TALENs and CRISPR/Cas9 not only outperform traditional genome engineering methods but also drastically reduce the cost and timeframe associated with generating targeted mutations. In addition to gene targeting in mice, TALENs and CRISPR/Cas9 can target organisms and cell types previously not amenable to such approaches including zebrafish, rats, drosophila, and C. elegans as well as human stem cells and iPSCs (induced pluripotent stem cells). Attesting to its usefulness, genome editing with engineered nucleases was named “Nature Method of the Year” in 2011.
Knock-Out and Knock-In Gene Targeting Vectors
Gene targeting vectors introduce mutations into endogenous loci by homologous recombination in ES cells. Mutation strategies include deletion of functional domains or translational start sites, introduction of frame shifts that terminate translation, deletion of entire genes, generation of point mutations or insertion of reporters or epitope tags.
Conditional gene modifications use the site-specific recombinase systems Cre-loxP or Flp-FRT to irreversibly inactivate (or activate) gene function in tissue or cell-type specific manners. Conditional approaches overcome the embryonic lethality associated with some germline null mutations or over-expression of a gene of interest. Gene expression is controlled by the interaction of Cre or Flp recombinase with a “floxed” or “frted” target gene using a dual transgenic mouse system.
Inducible systems allow for temporal and spatial control of gene expression. Tetracycline or tamoxifen regulated systems are commonly used to turn gene expression off or on. Inducible systems can be used in conjunction with conditional systems for an additional level of control.
Multifunctional vectors are designed to generate insertions and conditional deletions within the same gene.
Host sequences surrounding the transgene integration site can modify transgene expression; causing it to be weak, undetectable, or expressed in only a subset of expected cells or tissues. BAC transgenes can be used to ensure position-independent expression of the gene of interest by providing all regulatory sequences needed on a large (~150 – 200kb) segment of genomic DNA.
Promoter transgenes drive expression of a reporter or other gene of interest in a tissue or cell-type specific manner. Promoter transgenes integrate in random positions within the genome.
An initial consultation with the principal investigator is arranged to discuss the project strategy. After approval of the final construct design by the investigator and completion of a project submission form, the core will construct the vector and prepare DNA or RNA for microinjection or cell transfection.
Acknowledging the Core
Please acknowledge the Core in manuscripts as well as posters and talks. Suggested language: “This work was supported by the Hope Center Transgenic Vectors Core at Washington University School of Medicine.”
TALENs and CRISPR/Cas9 vectors
Initiating Use; User Fees
Please contact us to initiate a project, or inquire about transgenic vectors or custom services.
NOTE: Beginning July 2015, users must provide a fund number when a project is initiated.
Fees for Transgenic Vectors Core services are based on individual projects, and are subsidized by the Hope Center. This subsidy will apply only if the billing PI is a Hope Center faculty member.