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RNAi

RNAi. C. elegans and siRNA Penetrance and Expressivity. transcription. Alt. Splicing Alt. Poly-A, Alt. Translation Start. translation. Central Dogma addendum. DNA. RNA. Transcription Factors. Protein. TGS. transcription. PTGS. translation. Central Dogma addenda. DNA.

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RNAi

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  1. RNAi C. elegans and siRNA Penetrance and Expressivity

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

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

  8. 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.

  9. 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.

  10. RNAi

  11. delivery

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

  13. 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

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

  15. 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

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

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

  18. Pentrance? Expressivity?

  19. Pentrance? Expressivity?

  20. Pentrance? Expressivity?

  21. Pentrance? Expressivity?

  22. To Do • Tuesday: • Old: score hfr plates, • New: plate worms on dsRNA seeded plates, as well as plate controls, • Ongoing: Wasps.

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