Epigenetics: Nuclear transplantation & Reprogramming of the genome

Historical perspectives Big question: ‘nuclear cloning’ is possible? (genome of differentiated cells is identical to that of undifferentiated cells?) New concept: ‘Reprogramming’ (somatic to embryonic epigenetic state) Earlier studies with amphibian (frog eggs) Later studies with mammals (mouse eggs)

Nuclear transfer procedure (frog)

Nuclear transfer procedure (mouse) Earlier attempts: zygotes + nuclei from cleavage stage donor embryos Some success in farm animals not in mouse : lambs Transition time difference: maternal to zygotic transcription ‘Dolly’ cloning: mammary gland donor 1st somatic cell nuclear transfer (SCNT) More than 15 mammalian species cloned

Phenotype of cloned animals More differentiated-stage cells derive much less success! Frog nuclear transfers were successful up to tadpole stage!

Phenotype of cloned animals The majority fails after implantation G0 or G1 –phase donor cells are more successful upto blastocyst than S-phase ES or EC cells. But more success rates in ES or EC beyond the blastocyst stage. Cloned animals (survivors) likely have some defects that are responsible for adult-stage health problems. Large offspring syndrome (large fraction of placenta-specific genes are changed in terms of their expression levels) Gametogenesis reprogramming is important for placenta genes

Reprogramming of terminally differentiated cells (monoclonal mice) A little loss of genomic complement is not a problem for cloning!

Reprogramming of terminally differentiated cells (olfactory neuron)

Medical implications of nuclear transplantation Reproductive cloning vs Therapeutic cloning

Medical implications of nuclear transplantation (demonstration)

iPS (Induced Pluripotent Stem) cells three factors (Oct4, Sox2, Klf4) can transform adult cells into stem cells. does not require early-stage embryos or cells from patients

iPS (Induced Pluripotent Stem) cells