RNAi

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RNAi

• C. elegans and siRNA
• Penetrance and Expressivity

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Central Dogma addendum
DNA
RNA
Transcription Factors
Protein
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Central Dogma addenda
DNA
TGS Transcriptional Gene Silencing
RNA
Transcription Factors
Protein
PTGS Post Transcriptional Gene
Silencing
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tiny RNAs (20-to-25 nt RNAs)

• in eukaryotic cells, tiny RNAs function as
  transcriptional regulators of gene expression in
  (at least) three distinct pathways,
• (1) micro (miRNAs) regulate RNA translation.
• (2) small interfering RNAs (siRNAs) direct RNA
  destruction via the RNA interference (RNAi)
  pathway,
• (3) and transcriptional regulation via epigenetic
  processes,

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Ancient History (co-suppression)

• Transgene expression often decreases as the copy
  number of transgenes increased.

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Co-suppression Modes

• ...Transcriptional Gene Silencing (TGS),
• RNA functions in the methylation of promoters and
  structural elements of genes,
• ...Post-Transcritional Gene Silencing (PTGS),
• involves the specific degradation of mRNA via a
  double-stranded RNA intermediate, dsRNA.

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MoreModern
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RNAiRNA interference

• ...while attempting to do anti-sense KO of gene
  expression in C. elegans, Guo and Kemphues, Cell
  81, 611 (1995) observed that sense and anti-sense
  strands worked equally at reducing transcript,
• in an anti-sense experiment, a gene is
  constructed so that it produces a complementary
  strand to an expressed transcript,
• the goal is to complement, thus inactivate the
  mRNA.
• ...following up, other researchers found that
  dsRNA worked at least an order of magnitude
  better that either sense or anti-sense strands.

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RNAi

• ...siRNA control of gene expression by RNA
  processing is now considered a common element in
  eukaryotic cells,
• defense against viruses,
• control of transposable elements,
• regulate gene expression?

• useful for doing Reverse Genetic studies,
• dsRNA triggers sequence specific degradation of
  complementary mRNAs.

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RNAi
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C. Elegans and siRNA

• Bacteria expressing dsRNA (via plasmid usually)

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Genetic Stocks
Dumpy
Blister

• E. Coli Strains
• HT115(DE3)/pL440(bli-1) - blister
• HT115(DE3)pL440(dpy-11) - dumpy
• HT115(DE3) - empty RNAi feeding vector
  (control)
• C. elegans Strains
• dpy-11(e224)V Dumpy reference strain
• bli-1(e769)II Blister reference strain
• rrf-3(pk1426)II RNAi-sensitive worms, WT,
  N2

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Penetrance

• the frequency at which individuals with a given
  genotype manifest a specific phenotype.

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Penetrance

• 4 of 6 dogs, or 66 of the population shows the
  phenotype, at some level,
• penetrance is usually referred to as a
  percentage.

all the same genotype
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Expressivity

• the degree, or range in which a phenotype of a
  specific genotype is expressed.

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Expressivity

• range of phenotypes
• expressivity may be referred to as a percentage,
  or another quantifiable measure.

all the same genotype
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Pentrance? Expressivity?
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Pentrance? Expressivity?
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Pentrance? Expressivity?
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Pentrance? Expressivity?
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To Do

• Wednesday
• Score HFR plates, analyze data,
• Score Mutagenesis plates, analyze data,
• start lac- ? lac overnights.
• Thursday
• Plate overnight culture on lac selective media.
• Friday plate worms on dsRNA seeded plates, as
  well as plate controls,
• Score Lac revertants
• Ongoing Wasps!