uninjected, mex-3 probe

1. uninjected, no probe uninjected, mex-3 probe double-stranded mex-3 RNA injected, mex-3 probe antisense mex-3 RNA, mex-3 probe Double-stranded RNA-induced RNA interference causes destruction of a specific mRNA in C. elegans Guo, S. and Kemphues, K. J. Cell81, 611-620 (1995) Fire, A. et al. Nature391, 809 (1998)

2. Key points of C. elegans experiment • substoichiometric amounts of dsRNA relative to the targeted mRNA are • required to completely eliminate the mRNA (i.e. the dsRNA is catalytic) • dsRNA is 10-100X better than antisense or sense RNA • doesn’t work if introns or promoters are targeted by the dsRNA • doesn’t interfere with transcription initiation or elongation (it is possible • to target a single gene in an operon) (i.e. RNAi is a post-transcriptional • phenomena) • the targeted mRNA is degraded (i.e. it can’t be detected by probes) • dsRNA can cross cellular boundaries (i.e. there is a transport mechanism)

3. Mechanism of RNAi post-transcriptional gene silencing dsRNA 5’ small interfering RNAs 5’ (inactive, 250-500kDa complex) (a critical step in the activation of RISC) RNA-induced silencing complex (active, 100kDa complex) (endonucleolytic cleavage in the region of homology) Zamore, P. D. Science296, 1265-1269 (2002)

4. Dicer • Dicer contains 5 domains • 2 catalytic RNase domains • a dsRNA-binding domain • a helicase domain • a PAZ domain Dicer is thought to work as a dimer One of the catalytic sites in Dicer is defective Thus, instead of cleaving from ~9-11 nt, like bacterial RNase III Dicer cleaves ~22 nt (see panel B) Hannon, G. J. Nature418, 244-251 (2002)

5. RISC • RISC contains at least 4 subunits • Argonaute (5 homologs in Dros.) • dFXR (the Dros. homologue of • human fragile X mental retardation • protein) • Vasa intronic gene (VIG) • nuclease • Activated RISC uses the unwound • siRNA as a guide to substrate • selection Hannon, G. J. Nature418, 244-251 (2002)

6. Unanswered question • How does RNAi spread throughout and organism, even when triggered by • minute quantities of dsRNA? • -Requires a system to pass a signal from one cell to another • -Requires a strategy for amplifying the signal

7. RNAi can silence gene expression through other mechanisms: Histone methylation occurs at loci homologous to the siRNA target histone methyltransferase RNA-dependent RNA polymerase Matzke, M. and Matzke, A. J. M. Science 301, 1060-1061 (2003) Schramke, V. and Allshire, R. Science301, 1069-1074 (2003)

8. Questions about RNAi • What is the endogenous biological function of the RNAi machinery? • Gene Regulation • Protection from viruses. • It could to silence transposons • Is RNAi under negative regulation? • how about RNases that digest siRNAs • What are neurons refractory to RNAi?

17. mammals

18. cut A cut B T7 SP6 plasmid 3’ 3’ 5’ 5’ 5’ 5’ 3’ 3’ Synthesis of double-stranded RNA (dsRNA) in vitro cut A, SP6 polymerase, NTPs cut A SP6 T7 cut B, T7 polymerase, NTPs SP6 T7 cut B complementary 21-mer RNA oligonucleotides anneal 5’-GGCAAGGUCCAGCUGAACU-3’ ||||||||||||||||| 3’-UACCGUUCCAGGUCGACUU-5’ 800-1200 bp dsRNA

19. diploid and polyploid species of Chrysanthemum

24. Plant callus and cell culture Agrobacterium (and Rhizobium) mediated transformation Direct DNA transformation Plant virus vectors Gene transfer to plants

25. Somatic plant cells have high developmental plasticity (in contrast to most animal cells)--it’s as if they are all embryonic stem cells Cells from most parts of a plant can regenerate an entire new plant (totipotent) Plant cells can be cultured in suspension, genetically manipulated, then used to generate transgenic plants

26. Plant transgenics have been used to engineer: • Insect resistance (Bacillus thuringiensis [Bt] toxin) • Microbial pathogen resistance (e.g. overexpression of naturally-occurring plant defense genes, anti-fungal peptides “defensins” • Herbicide tolerance (glyphosate [Roundup] resistance gene) • Improved nutritional value (“golden rice” containing 1 bacterial and 2 daffodil genes for Vitamin A production).

27. “explant” (tissue sample) taken, disinfected With correct balance of plant hormones, a “callus” will form Callus transferred to liquid medium and agitated to yield a cell culture Single cells can be grown on plates to yield new calluses Plant tissue culture

28. Change phytohormone levels to get differentiation High auxin, roots develop High cytokinin, shoots develop In both cases plant formation can be induced OR Induce somatic embryogenesis: under specific conditions cells produce embryo-like structures that can develop into fertile plants Regeneration of plants from cell culture

29. Infection by Agrobacterium tumefaciens and its relatives Chemical transformation of protoplasts (cells lacking cell walls) Particle bombardment Viral infection Plant cell transformation

30. Plant tumors (crown gall disease) induced by bacterium Agrobacterium tumefaciens Tumors induced by Ti plasmids (140-235 kb) transferred to the plant cells by bacterium (Ti, Tumor inducing plasmids) Part of plasmid (T-DNA, 23 kb) integrates into plant genome (randomly): confers unregulated growth (hence a tumor) and directs synthesis of “opines” Opines provide food (carbon and nitrogen source) for bacterium, tumor provides a home, what a deal Agrobacterium transformation

31. A typical Ti plasmid • Virulence genes are responsible for T-DNA transfer--induced following bacterial attachment to plant wound • T-DNA is flanked by “border sequences” (25 bp imperfect direct repeats) involved in the transfer process: the right-hand border sequence is sufficient for transfer • T-DNA is excised by Vir gene products and transferred to plant cell via a conjugative pilus

32. Encode phytohormones to promote unregulated growth (oncogenes) These oncogenes can be deleted to “disarm” the T-DNA Disarmed T-DNA sequences are used for transformation T-DNA genes

33. An early ‘disarmed’ Ti plasmid Transfer could be screened for by opine synthesis (product of the nos gene)

34. Very large--difficult to manipulate in vitro Transgene-containing T-DNA can be created by recombination in Agrobacterium: Manipulating the Ti plasmid

35. T-DNA

36. Alternative approach: use two plasmids One with genes for virulence (“helper” plasmid) The other with T-DNA sequences--smaller, can use classical cloning techniques Manipulating the Ti vector

37. Drug resistance (e.g. aminoglycoside antibiotics) Herbicide resistance (e.g. glyphosate [Roundup]) Concern over potential harm (to health and/or environment) from these markers has driven development of other methods manA gene: confers growth on the sugar mannose as a sole carbon source Use of cre-lox mediated deletions to remove markers from transgenic plants Selection for T-DNA transfer

38. Agrobacterium-mediated transformation Kanamycin: selection for T-DNA transfer Carbenicillin: kills Agrobacterium

40. New developments: not just Agrobacterium can transfer genes via a T-DNA vector Closely related bacteria (Rhizobium species) can do the same This circumvents many patents, and researchers behind this work have made the technology freely available www.bioforge.net, see the “Transbacter” section

41. Fig. 10.25. Genetically engineered rice containing a biosynthetic pathway for beta-carotene

42. Protoplast transformation Make protoplasts (plant cells lacking cell walls) Addition of DNA in the presence of polyethylene glycol Electroporation Transformants selected on the basis of marker genes Regenerate whole plants Direct DNA transfer to plants

43. Particle bombardment Particles coated with transforming DNA, fired through plant cells (and nuclei) Valuable method for transforming plants that cannot be transformed by previous methods Works with cell cultures, embryos, leaves, etc. Direct DNA transfer to plants

44. Many chloroplasts in each cell (high copy number and expression of transgene) Chloroplasts are not transmitted by pollen (easier to contain the transgene) Chloroplast transgenes are not subject to position effects on transgene expression Target integration into chloroplasts using chloroplast homology regions Chloroplast transformation

45. Naturally transforming High-level transgene expression No integration into host chromosome Mostly used for expression of foreign proteins Plant viruses as vectors • DNA viruses • RNA viruses • Most plant viruses are RNA viruses • cDNA copies of the viral genome are used for engineering

46. Terminator technology Protection of technology and market share: “technology protection” (Monsanto) “terminator technology” (critics) Farmers historically save a small proportion of seeds from this year’s crop for next year’s crop Transgenic seeds: buy them once, never buy them again because of saved seeds Unless those transgenic plants produce sterile seeds

47. How to produce sterile seeds on demand: Ribosome inactivating protein (RIP) under the control of a late embryonic development induced promoter, remove transcription terminator by Cre/loxP system Plants produce viable seeds x RIP stop loxP loxP RIP No viable seeds (the seeds are soaked in Tet before sale)

49. Syngenta and the release of Bt-10 Bt-11: approved by govt. for sale to farmers Bt-10: not approved… differs from Bt-11 in that it has a marker gene (conferring ampicillin resistance) $375,000 fine for Syngenta Embarrassment (damage?) to the US biotech industry

50. How will the transgene affect: Ecosystem health a. Will the plant be more invasive? b. Will the plant transgenes transmit to the native plant population c. Will the plant harm beneficial animals and insects? d. How quickly will resistant strains of pathogens arise? Human health is recombinant DNA-containing plant tissue safe to eat over the long term? 3. Economics: will the choice to not grow transgenics harm farmers? Issues with recombinant plants