Transcript Control of Gene Expression - Washington State University

Control of Gene Expression
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
For some animals, a large library of
mutants has accumulated
• Drosophila
• C. elegans
• Mouse
animals with targeted mutations – gene knockout
1. Introduce DNA fragment containing altered version of target gene into
embryonic stem cells (ES cells) growing in culture
2. Separate cells and allow each to form a colony
3. Select those colonies in which the introduced fragment has replaced the
target gene
4. Inject ES cells into 3-day blastocysts to create chimeric embryos
5. Implant embryos into recipient mothers
6. 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
7. 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
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.
There is another way: gene
knockdown
• Two methods:
– antisense RNA
– inhibitory RNA
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
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
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
RNA inhibition is a naturallyoccurring 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.