1. C. elegans and siRNA Penetrance and Expressivity
RNAi
C. elegans and siRNA
Penetrance and Expressivity

2. Central Dogma DNA RNA Protein transcription translation addendum
Alt. Splicing
Alt. Poly-A,
Alt. Translation Start
Transcription
Factors
translation
Protein

3. Central Dogma DNA RNA Protein TGS: Transcriptional Gene Silencing TGS
addenda
DNA
TGS: Transcriptional
Gene Silencing
TGS
transcription
RNA
PTGS
Transcription
Factors
translation
Protein
PTGS: Post Transcriptional
Gene Silencing

4. 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,

5. Ancient History (co-suppression)
Transgene expression often decreases as the copy number of transgenes increased.

6. 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.

7. More Modern

8. 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 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.

9. RNAi …useful for doing Reverse Genetic studies,
...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.

10. RNAi

11. C. Elegans and siRNA Bacteria expressing dsRNA (via plasmid usually)…
Isopropyl β-D-1-thiogalactopyranoside
Tetracycline: bacteria
Ampicillin: plasmid (w/dsRNA)

12. 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

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

14. 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

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

16. Expressivity range of phenotypes
expressivity may be referred to as a percentage, or another quantifiable measure.
all the same genotype

17. Pentrance? Expressivity?

18. Pentrance? Expressivity?

19. Pentrance? Expressivity?

20. Pentrance? Expressivity?

21. To Do Ongoing: Wasps! 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!