Control of Gene Expression

1. Control of Gene Expression

2. Ways to study protein function by manipulating gene expression • Mutations • Naturally occurring, including human and animal genetic diseases • Induced by radiation or chemical mutagenesis • targeted • Knock-out animals –in which a particular gene of interest is replaced with a gene engineered to be non-functional • Gene knockdown – in which the mRNA for a particular gene is interfered with or destroyed

3. For some animals, a large library of mutants has accumulated • Drosophila • C. elegans • Mouse

4. animals with targeted mutations – gene knockout • Introduce DNA fragment containing altered version of target gene into embryonic stem cells (ES cells) growing in culture • Separate cells and allow each to form a colony • Select those colonies in which the introduced fragment has replaced the target gene • Inject ES cells into 3-day blastocysts to create chimeric embryos • Implant embryos into recipient mothers • Resulting 1st generation animals will be chimeric – some of them will have gonads formed from the introduced cells, so will be able to pass the altered gene on • Breed the 1st generation to one another to get transgenic animals in which the altered gene is present on both chromosomes – these are called knockout animals if the replacement gene is non-functional

5. Disadvantages of the knockout approach • The target protein may be so essential that it is backed up by other proteins (I.e., there is redundancy), so the phenotype shows no impairment. • Animals lacking the target gene may not survive embryonic development – this can now be overcome by making conditional knockouts – in which the gene of interest is only expressed in certain tissues or can be induced by defined stimuli • Making knockout animals is a time-consuming and expensive process – about 3 months and $200,000 for each knockout mouse line.

6. There is another way: gene knockdown • Two methods: • antisense RNA • inhibitory RNA

7. Antisense RNA • is single-stranded RNA complementary to the mRNA of the gene of interest • Cells take up the antisense RNA, which pairs with the native mRNA, interfering with its translation to protein

8. RNA interference (RNAi) • Generate doublestranded RNA complementary to the sequence of the mRNA of interest – cells take these up – or a viral vector may be needed to get them in. • An intracellular enzyme called Dicer cuts the dsRNA into small double and single-stranded fragments of 20-25 base pairs • Fragments associate with an RNA-induced silencing complex (RISC) composed of several proteins. This complex recognizes the mRNA containing the complementary sequence. One of the proteins in the complex (argonaute) then cleaves the mRNA into pieces, preventing its translation. • http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

9. Disadvantages of RNA-based gene knockdown • It’s usually temporary • Delivering the RNA may be problematic, depending on the organism –sometimes it is necessary to infect the organism with a vector virus carrying the desired RNA sequence

10. RNA inhibition is a naturally-occurring process • The genome contains a large amount of what was once characterized as “junk DNA” – we no realize that some of this codes for RNAi • The process is based on endogenous enzymes, which must have some natural function • It is also involved in viral disease, since some viral genomes code for RNAi that is effective against host cells • It is likely that RNAi will be a route of gene therapy.