EpigeneticsBroadly speakingTechnicallyIn other words“Cloning”: hello, Dolly, and hello again, DollySlide 6Slide 7?How can one explain the fact that cloning works so much better if one use a cell from an early embryo as the donor of the nucleus?Two explanationsSlide 11Slide 12Slide 13Slide 14Slide 15Slide 16DollySlide 18This cannot be!!! (except it is)ProofMonoclonal mice generated by nuclear transfer from mature B and T donor cellsEmbryonic stem cellsSlide 23Slide 24Slide 25Slide 26What they didThe resultImportant pointSemanticsExtensive abnormalities in cloned animalsProof that these abnormalities are entirely epigeneticNoteSlide 34Slide 35Slide 36Mice cloned from olfactory sensory neuronsNatalie Angier Unnatural ObsessionsSlide 39Sense of smellAllelic inactivation regulates olfactory receptor gene expressionSlide 42Slide 43Slide 44Question answerSolter and SuraniSlide 47The Haig hypothesisPeromyscus polionotus (the monogamous mouse)Slide 50The Barr bodyX-chromosome inactivationSlide 53Mechanism?Slide 55Slide 56Slide 57Slide 58Slide 59Histone methylationCalling David Duchovny and Gillian AndersonAh, plantsSlide 63No commentSlide 65Suppressors of clark kentkyp (kryptonite) codes for an H3K9 histone methyltransferase!Nature Genetics, Feb. ‘07Slide 69Slide 70Slide 71Slide 721MCB 140, 2/14/07EpigeneticsBeyond Mendel, part II2MCB 140, 2/14/07Broadly speakingAn “epigenetic” effect on the genome changes the phenotype without changing the genotype. The power of the environment and of life history3MCB 140, 2/14/07Technically“A mitotically or meiotically heritable change in gene expression state (or genome functional state) that is not associated with a change in the primary sequence of DNA.”4MCB 140, 2/14/07In other wordsGeneticsOrganism (or a cell) with a phenotype↓Mutation (change in DNA)↓Different phenotypeEpigeneticsOrganism (or a cell) with a phenotype↓Something happens, but not a change in the DNA↓Different phenotype5MCB 140, 2/14/07“Cloning”:hello, Dolly, andhello again, Dolly6MCB 140, 2/14/07King and Briggs (1956). Serial transplantation of embryonic nuclei Cold Spring Harbor Symp. Quant. Biol. 21: 271-289.7MCB 140, 2/14/07McKinnell, R. G. 1978. Cloning: Nuclear Transplantation in Amphibia. University of Minnesota Press, Minneapolis.8MCB 140, 2/14/07?9MCB 140, 2/14/07How can one explain the fact that cloning works so much better if one use a cell from an early embryo as the donor of the nucleus?10MCB 140, 2/14/07Two explanations1. Alteration of the actual DNA of the cells as the embryo develops.2. Something else.11MCB 140, 2/14/0712MCB 140, 2/14/0713MCB 140, 2/14/0714MCB 140, 2/14/0715MCB 140, 2/14/07Reya, Clarke, and Weissman. Nature 2001.16MCB 140, 2/14/07King and Briggs (1956). Serial transplantation of embryonic nuclei Cold Spring Harbor Symp. Quant. Biol. 21: 271-289.17MCB 140, 2/14/07Dolly18MCB 140, 2/14/07Ian Wilmut DollyBill Ritchie19MCB 140, 2/14/07This cannot be!!!(except it is)Maybe – maybe! – the cell that gave the nucleus that Dolly came from was not an actual mammary gland cell but rather an adult stem cell?20MCB 140, 2/14/07ProofHow – oh how – can one prove that the cell that one uses as a donor in nuclear transfer is a mature cell type?!!Well, in an ideal scenario, the nucleus would carry a signature of the fact that it came from a mature cell.But – but!! – all cells in the body have identical DNA.Or DO THEY?????21MCB 140, 2/14/07Monoclonal mice generated by nuclear transfer from mature B and T donor cellsHochedlinger K, Jaenisch R.Nature (2002)415:1035-1038.22MCB 140, 2/14/07Embryonic stem cells23MCB 140, 2/14/07Annu. Rev. Cell Dev. Biol. 2001. 17:435-462. EMBRYO-DERIVED STEM CELLS: Of Mice and Men Austin G. Smith24MCB 140, 2/14/0725MCB 140, 2/14/0726MCB 140, 2/14/0727MCB 140, 2/14/07What they did1. Take mouse B cell and mouse T cell.2. Take its nucleus out.3. Make an ES line from that nucleus by somatic cell nuclear transfer (put nucleus into enucleated egg, let this develop into embryo, disperse the embryo to make ES cells).4. Make a mouse from that ES line by “tetraploid complementation.”28MCB 140, 2/14/07The resultA monoclonal mouse29MCB 140, 2/14/07Important point“The difficulty of reprogramming differentiated B and T cells is consistent with the notion that many previously cloned animals may actually be derived from the nuclei of rare somatic stem cells present in heterogeneous donor cell populations, rather than from differentiated cells as has been assumed.”30MCB 140, 2/14/07Semantics1. Reproductive cloning: make new organisms.2. Therapeutic cloning (aka “somatic cell nuclear transfer”): no organism made.31MCB 140, 2/14/07Extensive abnormalities in cloned animals•Lung failure•Liver failure•Obesity•Etc etcTwo problems:1. Cloning is incredibly inefficient.2. Of the animals that are born, many have severe defects.32MCB 140, 2/14/07Proof that these abnormalities are entirely epigeneticDolly’s lambs, and the offspring of all cloned animals, are normal.33MCB 140, 2/14/07NoteNot all epigenetic effects vanish during meiosis. In plants, exposure to radiation increases overall DNA damage repair levels epigenetically and heritably (up to 4 generations).Transgeneration memory of stress in plants. Molinier J, Ries G, Zipfel C, Hohn B. Nature. 2006 Aug 31;442(7106):1046-9 In a very limited sense, this represents an “inheritance of acquired characteristics” as per Lamarck. This is a very rare phenomenon.34MCB 140, 2/14/07Prof. Jay Hollick, MCB35MCB 140, 2/14/07Pl Pl’36MCB 140, 2/14/07Pl is Changed to Pl’“paramutation”!37MCB 140, 2/14/07Mice cloned from olfactory sensory neurons Eggan et al.(Rudolf Jaenisch and Richard Axel)Nature (2004)428(6978):44-938MCB 140, 2/14/07Natalie Angier Unnatural Obsessions“The adjective that scientists use to describe a well-wrought experiment is “elegant” – which means not only that it worked, but it worked in style.”39MCB 140, 2/14/0740MCB 140, 2/14/07Sense of smell“One particularly clear example of neuronal diversity is provided by the olfactory sensory epithelium. In the mouse, each of the 2,000,000 cells in the olfactory epithelium expresses only one of about 1,500 odorant receptor genes, such that the functional identity of a neuron is defined by the nature of the receptor it expresses. Thus, the sensory epithelium consists of at least 1,500 neuronal types. The pattern of receptor expression is apparently random within one of four zones in
View Full Document
Unlocking...