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7.013 4.13.07 Life More great student questions of the day…. Q: Are all cells that are pluripotent also stem cells? 7.013 A: No! Many early embryonic cells are pluripotent, 4.13.07 but do not self-renew, and eventually differentiate. Q: What is the difference between a progenitor How-to 2 and a stem cell? Cloning and Epigenetics A: Somewhat semantic. Progenitors do not self-renew (or only do so for a short time), whereas stem cells have long term self-renewing capacity. STEM Future of LIFE CELLS& CLONING PRIONS How-to 2 3D STRUCTURE IMMUNE VISUAL NERVOUS VIRUSES 1. Reproductive vs CELL SYSTEMS TYPE & therapeutic cloning PROBLEMS PROBLEMS POSITION BACTERIA FORMATION FORMATION How-to 1 REC. DNA STEPS CANCER BIOCHEM GENETICS MOL. BIO CELL BIO. START FOUNDATIONS 1 2 blastula inner cell mass (ICM) Reproductive plate inner cloning: cell mass whole organism Purves 19.6: Making AGRICULTURE mammalian colonies of cells FERTILITY ES cells (each from single cell) ORGANS each colony may form a stem cell line: can have different potencies 3 Obtaining pluripotent stem cells from an adult: REPAIR Pluripotent stem cells 2. Cellular versus nuclear potency Adult Adult stem cells “Therapeutic” cloning Inject nucleus from adult cell into egg, remove ICM from resulting embryo and plate Glass needle 4 Extract 5 Somatic Cell Nuclear donor Transfer/ circa 1963 nucleus Meiotic spindle micropipette Remove Donor nucleus egg inserted into chromosomes enucleated cell host donor cloned frogs Membrane heals Somatic cell nucleus in activated egg embryo e g 6 7 a r s o u e . n l o c D u Isolate . n single cells . culture 0% form . embryo . Isolate . ~100% form . normal embryo 500 cell single nuclei.transplant into frog embryo enucleated egg Cellular versus nuclear potency H. Sive MIT 2007 Decrease in potency of nuclei with age of donor Dolly (+ lamb): first mammal cloned by nuclear transfer 1997 See Purves 19.4 Microscope set up for nuclear transfer 8 9 Isolation of somatic cell nuclei Removal of egg chromosomes (metaphase plate) Kevin Eggan and Rudolf Jaenisch, Whitehead Institute/MIT Kevin Eggan and Rudolf Jaenisch, Whitehead Institute/MIT 10 Problems with animals derived from SCNT Injection of somatic cell nucleus into enucleated egg Kevin Eggan and Rudolf Jaenisch, Whitehead Institute/MIT 11 Clone problems: die early, organ failure incorrect gene expression Control NT pup 3. Epigenetic control of gene expression “Large Offspring normal cloned Syndrome” 12 13 How identical are identical twins? Concordance (= measure of shared traits) The kitten “CC” is a non-identical clone produced using somatic nuclear transfer from “Rainbow” (B) scleroderma 98% identical, 10% fraternal twins Due to DNA base sequence asthma 54% identical, 24% fraternal twins Genes in A and B are same, gene expression is not. DNA base sequence + something else = Epigenetics 14 15 DNA is packaged into chromatin (histones + other proteins) Purves 9.6 In DNA, cytosine can be replaced by 5-methylcytosine: this does not change the base sequence of the DNA 15a 16 In chromatin Embryonic cells DNA is wound around establish histone proteins histones DNA methylation Ac Ac patterns Histones must be removed or modified (by specific enzymes) transcription to activate transcription Somatic cells maintain DNA methylation cell type-specific prevents histone methylation removal/modification to Me Me Me Me patterns repress transcription DNA methylation represses transcription Methylation is heritable during life of animal H.Sive MIT 2007 Ac Ac Ac Gene expression control: chromatin structure is crucial! 19a Addition of acetyl groups 17 to histone proteins Gene (nucleus) “opens” chromatin to histone modification chromatin activate transcription For your interest, not exam material! DNA methylation structure DNA Methylation transcription represses transcription Me Me BECAUSE RNA splicing, stability Ac Ac Ac export to cytoplasm MeDNA binds proteins which translation HDAC HDAC Me Me Me Me de-acetylate histones BP BP to inhibit transcription protein trafficking/ Modification/stability Connecting DNA methylation and histone acetylation H.Sive MIT 2007 H. Sive MIT 2007 18 19 Imprinting = paternal or maternal-specific methylation Cell type 1 Cell type 2 controls chromatin structure and transcription Gene 1 Me Me Gene 1 promoter egg Me Me Me imprinted expressed not expressed promoter Me Gene 2 imprinted Gene 2 Me Me Me Me Me Gene 3 not expressed expressed not imprinted Gene 1 Gene 3 sperm Me Me imprinted not expressed expressed Me Me Gene 2 imprinted Stable, cell type-specific methylation patterns help regulate which genes are expressed Me Me Gene 3 not imprinted H.Sive MIT 2007 H.Sive MIT 2007 20 adult Cell type A Cell type B egg Cell type C zygote embryo + Cell type D Why cloned embryos are sperm new Me new Me abnormal and how to reprogram Cell type E Imprinted Embryonic nuclei Me patterns Me patterns Cell type F Adult methylation patternAdults Somatic do not normally reverMet patterns DNA Methylation (Me) patterns (andto gene e mactivity)bry changeonic during pat developmentterns H.Sive MIT 2007 21 22 Future isolation of autologous stem cells? Methylation patterns and reprogramming (reprog.) in cloned embryos Oct3/4, Sox2, Normal development Cloned embryos + Klf4, c-Myc = Zygote differentiated “competent” state abnormal >99% reprogramming pluripotent adult cells (transcription) stem cells- incomplete reprog. (unipotent) factors and form many tissues Embryo in embryo early genes active normal <1% (repopulation complete reprog. SCNT assay- GFP label) What are Adult reprogramming adult genes active H.Sive MIT 2007 factors? Takahashi and Yamanaka, 2006.
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