Course summary
This laboratory-based training course will provide a comprehensive overview and practical laboratory experience of the genetic manipulation of mammalian stem cells.Focusing on mouse ES and human iPS cells, genome informatics, recombineering, CRISPR designer nucleases, transposon technology and gene targeting will be covered in interactive demonstrations, discussions and lectures by international experts. Laboratory work includes the culture and transfection of mouse ES and human iPS cells, design and construction of gene targeting vectors, and the use of PiggyBac transposons and designer nucleases (CRISPRs) for advanced genome engineering and genetic screens. Participants will be trained in the informatics and practical use of public IKMC (International Knockout Mouse Consortium) mouse knockout resources, including the production of mutant mice.Emphasis is placed on the planning/design and successful experimental execution of gene targeting projects and participants should gain the skills necessary to design, construct and target their own genes of interest.
Course programme
1. Informatics The informatics underlying the visualization and interpretation of gene structures will be presented focusing on the practical design of gene targeting strategies. Students will also develop their own CRISPR-mediated gene targeting designs using web-based tools and learn how to exploit the public IKMC/IMPC mouse gene targeting/phenotyping resources. 2. Recombineering of BACs and Gene Targeting Vectors Students will use recombineering to make modified BAC transgenes and gene targeting constructs. The theoretical principles underlying both recombineering and rational targeting vector design will be presented in lectures and practical exercises. 3. Modular Gene Targeting Vectors Students will learn to apply modular approaches to vector assembly and gene targeting strategies.Students will build their own vectors for CRISPR-based gene targeting using Gibson assembly. 4. Stem Cell Culture/Gene Targeting Students will learn feeder-free culture of mouse ES cells and human iPS cells including a number of standard transfection techniques (electroporation, nucleofection, lipofection). Transfection, selection, picking, processing and genotyping for gene targeting in ES cells will be covered. 5. CRISPR Designer Nucleases A CRISPR mediated homologous recombination experiment will be performed in human IPS cells.Practical application of the CRISPR system to undertake in vitro genetic screens will also be explored. 6. Transposon Technology/IPS Cell Generation Students will be introduced to the uses of the highly efficient PiggyBac transposon systems for BAC transgenesis and reprogramming for iPS.Know more
0 comments:
Post a Comment