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RNAi
C. elegans and siRNA
Penetrance and Expressivity
Central Dogma
addendum
DNA
transcription
RNA
Alt. Splicing
Alt. Poly-A,
Alt. Translation Start
translation
Protein
Transcription
Factors
Central Dogma
addenda
DNA
TGS: Transcriptional
Gene Silencing
TGS
transcription
RNA
PTGS
Transcription
Factors
translation
Protein
PTGS: Post Transcriptional
Gene Silencing
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,
Ancient History
(co-suppression)
Transgene expression often decreases as the copy number of transgenes increased.
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.
More
Modern
RNAi
RNA 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 antisense 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.
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.
RNAi
C. Elegans and siRNA
• Bacteria expressing dsRNA (via plasmid usually)…
Isopropyl β-D-1-thiogalactopyranoside
Tetracycline: bacteria
Ampicillin: plasmid (w/dsRNA)
Genetic Stocks
Blister
Dumpy
• 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
Penetrance
…the frequency at which individuals with a
given genotype manifest a specific
phenotype.
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
Expressivity
…the degree, or range in which a phenotype
of a specific genotype is expressed.
Expressivity
• range of
phenotypes
• expressivity may be
referred to as a
percentage, or
another quantifiable
measure.
all the same genotype
Pentrance? Expressivity?
Pentrance? Expressivity?
Pentrance? Expressivity?
Pentrance? Expressivity?
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!