Control of Gene Expression

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Control of Gene Expression
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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

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For some animals, a large library of mutants has
accumulated

• Drosophila
• C. elegans
• Mouse

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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

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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
• Making knockout animals is a time-consuming and
  expensive process about 1 year and 200,000 for
  each knockout mouse line.

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There is another way gene knockdown

• Two methods
• antisense RNA
• inhibitory RNA

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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

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RNA interference

• Generate doublestranded RNA complementary to the
  sequence of the mRNA of interest cells take
  these up.
• 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

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Disadvantages of RNA-based gene knockdown

• Its 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

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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.