{"id":70,"date":"2020-07-31T20:21:55","date_gmt":"2020-08-01T00:21:55","guid":{"rendered":"https:\/\/pressbooks.bccampus.ca\/kathleef\/?post_type=chapter&p=70"},"modified":"2025-06-16T16:16:35","modified_gmt":"2025-06-16T20:16:35","slug":"chapter-9-investigating-gene-function-2-knock-out-and-knock-down","status":"publish","type":"chapter","link":"https:\/\/pressbooks.bccampus.ca\/kathleef\/chapter\/chapter-9-investigating-gene-function-2-knock-out-and-knock-down\/","title":{"raw":"Investigating Gene Function Part 3 - Knock-down and Knock-out","rendered":"Investigating Gene Function Part 3 – Knock-down and Knock-out"},"content":{"raw":"

Introduction<\/h1>\r\nIn this section we will look at ways to go beyond detecting when and where a gene is expressed; we will explore ways of reducing or eliminating the gene's expression, and using the resulting phenotype to deduce the likely role of the gene product. Understanding when and where the gene is expressed (discussed in Chapter 7<\/a> and Chapter 8<\/a>) will help us know where we should expect to see the phenotype resulting from reduced or no expression of the gene. It is an important first step in elucidating gene function.\u00a0 Note that we could be surprised, however. Sometimes despite the knowledge we have accumulated about a gene, we find it works in an unexpected way - and produces a phenotype we would not have predicted. This is what is exciting about science- we don't really know the answer until the experiments are done.\r\n\r\n \r\n\r\nWe will cover four means of eliminating or reducing gene expression here:\u00a0 insertional mutagenesis, homologous recombination, RNAi, and CRISPR-Cas9. There will be the strongest focus on the last two of these, both of which are borrowed from bacteria. We will consider how they work in nature, protecting cells from infection and then how they are used in analysis of gene function.\u00a0 The approaches used to make the constructs for these applications will also be described.\r\n\r\n \r\n
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Contents<\/h2>\r\n

Learning Outcomes<\/a>\r\nTerminology before we begin<\/a>\r\nA. Insertional Inactivation<\/a><\/p>\r\n

A-1. Ti Plasmids in Plants<\/a>\r\nA-2. P elements in fruit flies<\/a><\/p>\r\n

B. Homologous Recombination<\/a><\/p>\r\n

C. RNAi<\/a><\/p>\r\n

C-1. A little history<\/a>\r\nC-2. How it works<\/a>\r\nC-3. Making constructs for RNAi<\/a><\/p>\r\n

D. CRISPR<\/a><\/p>\r\n

D-1. Preamble<\/a>\r\nD-2. How bacteria do it<\/a>\r\nD-3. How we do it: making CRISPR constructs<\/a><\/p>\r\n

D3-i. Knock out - NHEJ repair<\/a>\r\nD3-ii. Directed modification - HDR<\/a>\r\nD3-iii. Designing oligonucleotides that will generate an overhang for cloning<\/a><\/p>\r\n \r\n\r\nList of lecture videos (excluding supplemental videos)<\/a>\r\n\r\n<\/div>\r\n\r\n

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<\/a>Learning Outcomes<\/h2>\r\n<\/header>\r\n
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