Mouse Genetics Group
The Mouse Genetics Group was started in 2007 to provide a forum for researchers to share ideas, experimental approaches, and resources, while providing opportunities for labs wishing to develop transgenic mouse models to connect with the broader community. The Mouse Group is led by Raj Awatramani, assistant professor of neurology at Feinberg, and Tom Bozza, assistant professor of neurobiology and physiology at Weinberg College, and is supported by the Center for Genetic Medicine.
Fall 2009 Group Meeting
Tuesday, October 6, 2009
2:00-3:30 pm
Baldwin Auditorium
Robert H. Lurie Medical Research Center
303 East Superior Street
Chicago, IL 60611
"Targeted Genome Editing in Mammalian Cells using Engineered Zinc Finger Nucleases"
Aron Geurts, PhD, Assistant Professor of Physiology, Medical College of Wisconsin
Detailed physiological and genetic characterizations of the rat have proven that it is a robust model for the dissection of human disease-related traits. The ability to generate site-directed mutations to generate new disease models and to validate genes within quantitative trait loci would be welcome, particularly if it was widely applicable to commonly used strains. While the isolation of germline-competent rat embryonic stem cells as well as induced pluripotent stem (IPS) cells has recently been accomplished from a limited number of rat strains, gene targeting in these cells has yet to be described. As an alternative approach, we can now demonstrate that rat genes can be efficiently modified in the embryo directly after a single microinjection of designed zinc-finger nucleases (ZFNs). Using ZFNs designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M and Rab38, we demonstrate that a single injection of DNA or mRNA encoding ZFNs into the 1-cell rat embryo leads to a high frequency of animals carrying 25-100% disruption at the target locus. Importantly, these mutations faithfully and efficiently transmitted through the germline. Our data demonstrate that ZFN are highly active during the earliest cell divisions in rat embryos leading to whole-animal monoallelic and, on occasion, biallelic gene disruption with no observable off-target effects. Our data demonstrate, for the first time, the feasibility of generating knockout rat models on multiple strain backgrounds with unprecedented speed, paving the way to engineering the next generation of human disease models.
Jeffrey Porter, Field Application Specialist, Sigma-Aldrich
Rational genome engineering in mammalian cells is of enormous potential across basic research, drug-discovery as well as cell-based medicines. To this end, Sangamo Biosciences and Sigma-Aldrich have recently partnered to commercialize a novel technology that enables high0-frequency genome editing via the application of designed zinc finger nucleases (ZFNs) Within these chimeric proteins the DNA binding specificity of the zinc finger protein determines the site of nuclease action. Such engineered ZFNs are able to recognize and bind to a specified locus and evoke a double-strand break (DSB) in the targeted DNA with high efficiency and base-pair precision. The cell then employs the natural DNA repair processes of either “homology-directed repair (HDR)” or “non-homologous end joining (NHEJ)” to heal the targeted bread. These two pathways provide the investigator with the ability to provoke three unique outcomes in genome editing-gene correction, gene deletion and targeted gene addition. Furthermore, the speed and efficiency of this process enables us to knockout multiple genes in the same cell. Drawing from our work with transformed cell lines, primary human cells and multi-potent stem cells, we will present several examples of single, double and triple gene knockout in mammalian cells. We will also demonstrate use of ZFNs for rapid creation of a variety of knockout organisms, including zebrafish, mice and rats. Furthermore, we demonstrate the use of ZFN technology for targeted gene insertion into native chromosomal loci in cells including human and mouse embryonic stem cells.
Refreshments will be provided.
Contact Anna Shepherd at annashepherd@northwestern.edu with questions.
***Sponsored by the Center for Genetic Medicine of Northwestern University and Sigma Aldrich***
