Course on Genetic Manipulation of ES Cells, 5-18 November 2012
Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
Deadline for applications: 6 July 2012
This laboratory-based training course will provide a comprehensive overview and practical laboratory experience of the genetic manipulation of mouse ES cells for a broad range of applications. Recent advances in genome informatics, recombineering, transposon technology and uses of conditional gene targeting will be covered in lectures by both instructors and invited expert speakers and through interactive demonstrations.Laboratory work will focus on the culture and transfection of ES cells, design and construction of gene targeting vectors from BACs by recombineering, genotyping of gene targeting events and the practical use of transposon technology. Participants will also be trained in the informatics and practical use of public gene targeting resources being produced by the IKMC (International Knockout Mouse Consortium).
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)
The Course programme includes the following topics:
The informatics underlying the visualization of gene structures and the design of gene targeting vectors, recombineering oligos and genotyping primers will be demonstrated. Students will also run their own gene targeting designs using web-based tools.The informatics of locating existing public IKMC gene targeting resources will also be covered.
2. Recombineering of Gene Targeting Vectors
Students will build a variety of targeting constructs from BACs using recombineering technology. The theoretical principles underlying both recombineering and rational targeting vector design will be emphasized by both lectures and practical exercises.
3. ES Cell Culture
Students will learn feeder-dependent and feeder-free culture of ES cells derived from 129 and C57BL/6 mouse strains.ES cell colonies will be picked, expanded and frozen and subsequently thawed to test their integrity.
4. Gene Targeting in ES Cells
Students will electroporate conditional gene targeting constructs into ES cells and learn to genotype cells by LR-PCR and qPCR-based methods.
5. Transposon Technology
Students will be introduced to the uses of the highly efficient piggyBac transposon system for expression, mutagenesis and mouse induced pluripotent stem cell (iPS) cell generation.
6. Modular Targeting Resources
Students will 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.