March 10, 2014 All Day
This educational and lab-based training class will provide someone with a comprehensive overview and practical laboratory experience in genetic manipulation of mammalian stem cells.This course focuses on mouse ES and human iPS cells, recent advances in genome informatics, recombineering, custom nucleases (CRISPRs/TALENs), transposon technology and conditional gene targeting will be covered in lectures and through interactive demonstrations. Laboratory work includes the culture and transfection of Embryonic Stem cells, design and construction of gene targeting vectors by BAC recombineering, and the use of transposons and designer nucleases for advanced genomic engineering.
Students will be trained in the informatics and practical use of public gene targeting resources, including the production of mice from the International Knockout Mouse Consortium. Emphasis is placed on the planning/design and successful experimental execution of gene targeting projects.
The informatics underlying the visualization of gene structures and the design of gene targeting vectors, recombineering oligos and genotyping primers will be demonstrated. will also run their own gene targeting designs using web-based tools and learn how to use IKMC gene targeting resources.
2. Recombineering of BACs and Gene Targeting Vectors
will use recombineering to make BAC transgenes and targeting vectors. The theoretical principles underlying both recombineering and rational targeting vector design will be presented in lectures and practical exercises.
3. Stem Cell Culture
will learn feeder-free culture of ES cells derived from 129 and C57BL/6 mouse strains and human iPS cells.ES cell colonies will be picked, expanded and frozen and subsequently thawed to test their viability.
4. Gene Targeting in mouse ES Cells
Transfection, selection, picking, processing and genotyping for gene targeting in ES cells will be covered.
5. Transposon Technology
Learn uses of the highly efficient transposon systems for BAC transgenesis, insertional mutagenesis and reprogramming for iPS.
6. TALENs/CRISPRs – Site Specific Nucleases
You will transfect ES cells with TALEN/CRISPRs and learn about the applications of custom site-specific nucleases in stem cell genome engineering.
7. Modular Targeting Resources
Assemble a variety of modular knock-in targeting vectors from IKMC resources and analyse their integrity.Recombination Mediated Cassette Exchange (RMCE) using Flp and Cre recombinases will be used to modify IKMC conditional alleles directly in ES cells.
8. Homozygous ES Cells
Learn about approaches used to generate homozygous ES cells and the analysis of cellular phenotypes.
- Professor Francis Stewart (Dresden University of Technology, Dresden, Germany)
- Dr William Skarnes (Wellcome Trust Sanger Institute, Cambridge, UK)
- Dr Pentao Liu (Wellcome Trust Sanger Institute, Cambridge, UK)
- Dr Barry Rosen (Wellcome Trust Sanger Institute, Cambridge, UK)
- Professor Allan Bradley (Wellcome Trust Sanger Institute, Cambridge, UK)
- Dr Timm Schroeder (Helmholtz Zentrum München, Germany)
- Dr Ludovic Vallier (Wellcome Trust Centre for Stem Cell Research, Cambridge, UK)
- Dr Anton Wutz (Department of Biochemistry, University of Cambridge, UK)
- Professor Jin-Soo Kim (National Creative Research Initiatives Center for Genome Engineering, Seoul, South Korea)
- Dr Ralf Kuhn (Institute for Developmental Genetics, Munich, Germany)
- Dr David Frendewey (Regeneron Pharmaceuticals, Inc., USA)
- Dr Wolf Reik (Wellcome Trust Sanger Institute, Cambridge, UK)
- Professor Austin Smith (Wellcome Trust Centre for Stem Cell Research, Cambridge, UK)
- Dr Chad Cowan (Harvard Stem Cell Institute, Massachusetts, USA)
- Dr Kosuke Yusa (Wellcome Trust Sanger Institute, Cambridge, UK)
2014 Course on Genetic Engineering of Mammalian Stem Cells
March 10-22, 2014
Trust Genome Campus, Hinxton, Cambridge