Transgenic Vectors

Design and construction of TALENs, CRISPR/Cas9 and gene targeting vectors for genome engineering
Read More

Notice re Hope Center Subsidy

Beginning with the August 2013 billing cycle, the Hope Center subsidy will apply only if the billing PI is a Hope Center faculty member.

About

Neuromuscular Junction - Josh Sanes

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.

ChAT KO NMJ Synaptic Sites, Josh Sanes

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.

Transgenic Vectors Publications

Services

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

Synaptic Vesicles - Rachel Wong

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 Vectors

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.

Bipolar Cells, Rachel Wong

Inducible Vectors

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

Multifunctional vectors are designed to generate insertions and conditional deletions within the same gene.

BAC Transgenes
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

Retina, Rachel Wong

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.

Construct Request

Neuromuscular Junction, Josh Sanes

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.”

EmailPrintShare

TALENs and CRISPR/Cas9 vectors

More about TALENs and CRISPR/Cas9

.

Contact Information

Initiating Use

Please contact us to initiate a project, or inquire about transgenic vectors or custom services.

Contact the core