2016 Lab 4
Cell Knockout Methods
I apologise for this late notice. The research academic who was meant to run this practical class is now unavailable, and I now already have another teaching commitment between 11-1 pm tomorrow. I have been unable to find a replacement for this practical class. I will attempt to be present at the start of the class or sometime during the 2 hours when I get a break in my other workshop.
Dr Anthony Kee Slides - The following set of slides would have formed part of Dr Anthony Kee's 2016 presentation for this lab on Knockout Methods. There will also be an associated individual assessment based upon this lab.
DNA targeting platforms for genome editing
Lab 4 Individual Assessment
For Lab 4 class working in your Project Group, design an experiment employing CRISPR knockout technologies that would investigate a human disease.
The experiment should have:
- Hypothesis (the hypothesis you are testing)
- Aims (a series of specific aims of your experimental design)
- Method (the design of your KO experimental procedure, referenced)
- Results (how the results/outcomes could be interpreted/tested)
This exercise should be completed during the class timetable period and posted onto this page. 2016 Lab 4 - CRISPR/Cas9
See also video JoVE Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Some Recent Reviews
- Genome-editing Technologies for Gene and Cell Therapy. "Gene therapy has historically been defined as the addition of new genes to human cells. However, the recent advent of genome-editing technologies has enabled a new paradigm in which the sequence of the human genome can be precisely manipulated to achieve a therapeutic effect. This includes the correction of mutations that cause disease, the addition of therapeutic genes to specific sites in the genome, and the removal of deleterious genes or genome sequences. This review presents the mechanisms of different genome-editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, transcription activator-like effector nucleases (TALENs), meganucleases, and the CRISPR/Cas9 system. We then summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders. The current challenges and future prospects for genome editing as a transformative technology for gene and cell therapy are also discussed."
- Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old-new. "In the recent years, sequence-specific nucleases such as ZFNs, TALENs, and CRISPR/Cas9 have revolutionzed the fields of animal genome editing and transgenesis. However, these new techniques require microinjection to deliver nucleic acids into embryos to generate gene-modified animals. Microinjection is a delicate procedure that requires sophisticated equipment and highly trained and experienced technicians. Though over a dozen alternate approaches for nucleic acid delivery into embryos were attempted during the pre-CRISPR era, none of them became routinely used as microinjection. The addition of CRISPR/Cas9 to the genome editing toolbox has propelled the search for novel delivery approaches that can obviate the need for microinjection. Indeed, some groups have recently developed electroporation-based methods that have the potential to radically change animal transgenesis. This review provides an overview of the old and new delivery methods, and discusses various strategies that were attempted during the last three decades. In addition, several of the methods are re-evaluated with respect to their suitability to deliver genome editing components, particularly CRISPR/Cas9, to embryos."
- Morgan L Maeder, Charles A Gersbach Genome-editing Technologies for Gene and Cell Therapy. Mol. Ther.: 2016, 24(3);430-46 PubMed 26755333
- Masahiro Sato, Masato Ohtsuka, Satoshi Watanabe, Channabasavaiah B Gurumurthy Nucleic acids delivery methods for genome editing in zygotes and embryos: the old, the new, and the old-new. Biol. Direct: 2016, 11(1);16 PubMed 27037013
- Molecular Biology of the Cell 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002. Studying Gene Expression and Function
- Molecular Cell Biology 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000 Section 8.5 Gene Replacement and Transgenic Animals
- Developmental Biology 6th edition. Gilbert SF. Sunderland (MA): Sinauer Associates; 2000. Transgenic cells and organisms
Search Pubmed Databases: term=knockout mice
External Links Notice - The dynamic nature of the internet may mean that some of these listed links may no longer function. If the link no longer works search the web with the link text or name.
- The Nobel Prize Physiology or Medicine 2007 awarded jointly to Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies for their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells.
- NIH List of Available Knockout Mice | Factsheet
- International Knockout Mouse Consortium IKMC | Research using IKMC products
- NC3Rs Mouse databases
2016 Course Content
Lectures: Cell Biology Introduction | Cells Eukaryotes and Prokaryotes | Cell Membranes and Compartments | Cell Nucleus | Cell Export - Exocytosis | Cell Import - Endocytosis | Cytoskeleton Introduction | Cytoskeleton - Microfilaments | Cytoskeleton - Microtubules | Cytoskeleton - Intermediate Filaments | Cell Mitochondria | Cell Junctions | Extracellular Matrix 1 | Extracellular Matrix 2 | Cell Cycle | Cell Division | Cell Death 1 | Cell Death 2 | Signal 1 | Signal 2 | Stem Cells 1 | Stem Cells 2 | Development | 2016 Revision
Laboratories: Introduction to Lab | Microscopy Methods | Preparation/Fixation | Cell Knockout Methods | Cytoskeleton Exercise | Immunochemistry | Project Work | Confocal Microscopy | Tissue Culture | Stem Cells Lab | Microarray Visit
Dr Mark Hill 2015, UNSW Cell Biology - UNSW CRICOS Provider Code No. 00098G