Transcript RNAi
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, – 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-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 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 dsRNA seeded plates, as well as plate controls, – Ongoing: Wasps.