RNAi C elegans and si RNA Penetrance and

RNAi C. elegans and si. RNA 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 (mi. RNAs) regulate RNA translation. – (2) small interfering RNAs (si. RNAs) 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 m. RNA via a double-stranded RNA intermediate, ds. RNA.

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, – 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 m. RNA. . following up, other researchers found that ds. RNA worked at least an order of magnitude better that either sense or anti-sense strands.

RNAi. . . si. RNA 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, – ds. RNA triggers sequence specific degradation of complementary m. RNAs.

RNAi

delivery

C. Elegans and si. RNA • Bacteria expressing ds. RNA (via plasmid usually)… Isopropyl β-D-1 -thiogalactopyranoside Tetracycline: bacteria Ampicillin: plasmid (w/ds. RNA)

Genetic Stocks Blister Dumpy • E. Coli Strains: • HT 115(DE 3)/p. L 440(bli-1) - blister • HT 115(DE 3)p. L 440(dpy-11) - dumpy • HT 115(DE 3) - empty RNAi feeding vector (control) • C. elegans Strains: • dpy-11(e 224)V – Dumpy reference strain • bli-1(e 769)II – Blister reference strain • rrf-3(pk 1426)II – RNAi-sensitive worms

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 • Tuesday: – Old: score hfr plates, – New: plate worms on ds. RNA seeded plates, as well as plate controls, – Ongoing: Wasps.