RNAi

C. elegans and siRNA

Penetrance and Expressivity

Central Dogmaaddendum

DNA

RNA

transcription

Protein

translation

Transcription Factors

Alt. SplicingAlt. Poly-A,Alt. Translation Start

Central Dogmaaddenda

DNA

RNA

transcription

Protein

translation

Transcription Factors

TGS

PTGS

TGS: Transcriptional Gene Silencing

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.

MoreModern

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,

– 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

delivery

C. Elegans and siRNA

• Bacteria expressing dsRNA (via plasmid usually)…

Isopropyl β-D-1-thiogalactopyranosideTetracycline: bacteriaAmpicillin: plasmid (w/dsRNA)

Genetic Stocks

• 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

DumpyBlister

Penetrance

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

• 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

Penetrance

Expressivity

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

• range of phenotypes

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

all the same genotype

Expressivity

Pentrance? Expressivity?

Pentrance? Expressivity?

Pentrance? Expressivity?

Pentrance? Expressivity?

To Do

• Tuesday:

– Old: score hfr plates,

– New: plate worms on dsRNA seeded plates, as well as plate controls,

– Ongoing: Wasps.