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7.72
12.4.06
Cloning and Epigenetics
Model
organismsAnalysis Principles
Organ
formation
Human
issuesCloning
Stem
cells
Axon
guidance
Foundations
+ +
Developmental Readout
3D
structureAxes
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Cloning:
Making an identical copy
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Epigenetics:
Transcriptional control that does
not directly involve DNA base sequence,
and can be reversible without mutation
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1. Reproductive vs
therapeutic cloning
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Artisan, 1978
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Why clone?
Reproductive
replacement baby
infertility
spare parts
gene therapy
useful producer animal
Therapeutic
autologous stem cells
gene therapy
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Obtaining Human Pluripotent Stem Cells
from an adult
Therapeutic cloning
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2. History: the question of
cellular potency
Cells in 8 cell
sea urchin
are not
totipotent.
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3. Testing the potency of nuclei
Nuclear transfer method
for cloning frogs/ circa 1963
Remove
egg chromosomes
plus spindle
Glass needle
Meiotic
spindle micropipette
Membrane
heals
Extract
donor
nucleus
Donor nucleus
inserted into
enucleated cell
Somatic cell
nucleus
in an
activated egg
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host donor cloned frogs
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Decrease in potency of nuclei with age of donor
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500 cell
frog embryo
.
.
.
.
.
Isolate
single cells culture0% form
embryo
...
.
.Isolate
single nuclei transplant into
enucleated egg
~100% form
normal embryo
Cellular versus nuclear potency
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4. SCNT Methodology
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Dolly:
first mammal cloned by
nuclear transfer 1997
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Cloning mammals using adult somatic nuclei11a
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Cloning mammals using adult somatic nuclei
11b
Microscope set up for
nuclear transfer
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Isolation of somatic cell nuclei
Kevin Eggan and Rudolf Jaenisch, Whitehead Institute
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Removal of egg chromosomes (metaphase plate)
Kevin Eggan and Rudolf Jaenisch, Whitehead Institute
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Injection of somatic cell nucleus into enucleated egg
Kevin Eggan and Rudolf Jaenisch, Whitehead Institute
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5. Problems with animals derived
from nuclear transfer
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“Large Offspring Syndrome”
In Cloned Mice
Control NT pup
normal cloned
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Early death of mice cloned from somatic cells
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GPDH
Oct4
Dppa1
Prame14
Prame16
Prame15
Prame17
Ndp5211
Dppa2
Dppa3
Dppa4
Dppa5
ES cells
ES clone
morula
ES clone
blastocyst
Cumulus
morula
Cumulus
morula
Cumulus
blastocyst
Cumulus
blastocyst
Embryos
derived from
cumulus cells
do not show
normal early gene
expression patterns
Bortvin et al, 2003
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Individual cloned embryos are different
Bortvin et al, 2003
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Most nuclear transfers result in partial blastulae
Byrne et al, 2003
Potency of cells in partial blastulae?
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Normal muscle from abnormal cloned embryo
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6. Epigenetic control
of gene expression
The kitten “CC” is a non-identical clone produced
using somatic nuclear transfer from “Rainbow” (B)
Genes in A and B are same, gene expression is not.
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How identical are identical twins?
Concordance (= do both twins share a trait?)
scleroderma 98% identical, 10% fraternal
asthma 54% identical, 24% fraternal
Due to DNA base sequence
DNA base sequence + something else = Epigenetics
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DNA is packaged
into chromatin
Core nucleosome =
146bp DNA + core histones
H2A, H2B, H3 and H4
Linker histone = H1
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Kaeser and Emerson, 2006
Determination, differentiation and chromatin
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DNA Methylation
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In DNA, cytosine can be replaced by 5-methylcytosine:
this does not change the base sequence of the DNA
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Methylation of Globin Genes
represses their expression
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Cell type-specific methylation patterns
help regulate which genes are expressed
Cell type 1 Cell type 2
Gene 1
Gene 2
Gene 3
H.Sive MIT 2006
expressed
MeMeMe
not expressed
MeMeMe
not expressed expressed
expressed
MeMe
not expressed
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Another epigenetic control
paternal or maternal-specific methylation = imprinting
egg
spermGene 1
imprinted
Me MeGene 2
imprinted
Me Me Gene 3
not imprinted
Me Me Gene 3
not imprinted
Me Gene 1
imprinted
Me
Gene 2
imprinted
Me
H.Sive MIT 2006
29
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Santos and Dean, 2004
Global changes in paternal and maternal methylation
during mouse development
Red: maternal
Blue: paternal
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Germ cells
and embryonic cells
establish
methylation
patterns
Somatic cells
maintain
cell type-specific
methylation
patterns
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Klose and Bird, 2006
Targetting de novo DNA methylation via
DNA sequence recognition
32Direct DNMT recognition
TF/DNMT recognition
siRNA signal recognition
egg
sperm
zygote embryo+
Gene Methylation (Me) patterns
(and therefore gene activity) change during development
Imprinted
Me patterns
Primordial
germ cellsnew Me
de-Me
H.Sive MIT 2006
new Me
Embryonic
Me patterns
adult
Cell type A
Cell type B
Cell type C
Cell type D
Cell type E
Cell type F
Somatic Me
patterns
new Me
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Histone modification
and DNA methylation connection
“Histone code”.
Sites of histone
H3, H4
acetylation and
methylation
Grewal and Mozaed, 2003
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In chromatin
DNA
is wound around
histone proteins
Histone removal, and therefore transcription,
is inhibited by DNA methylationH.Sive MIT 2006
Methylation of cytosine
prevents histone
removal/modification and so
represses transcriptionMe Me
histones DNA
Histones must be
removed or modified
to activate transcriptiontranscription
dsDNA is shown as one line35
In chromatin
DNA
is wound around
histone proteins
Transcription requires acetylated histones and is
inhibited by DNA methylationH.Sive MIT 2006
Addition of acetyl groups
to histone proteins
unwinds chromatin to
activate transcription
Ac
Ac
Ac
Methylation of cytosine
represses transcription
Me Me
histones DNA
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DNA Methylation
inhibits transcription
DNA methylation directs histone de-acetylationH.Sive MIT 2006
Ac
Ac
Ac
Me Me
because…
Me MeMe
BP
Me
BP
HDACHDAC
MeDNA binds specific
proteins
Me
BP
Me
BPwhich bind
Histone Deacetylase
(HDAC)
HDACHDAC
Ac
Ac
Ac
Me Me
which…
de-acetylates histones
are therefore inhibits
transcription
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Multiple mechanisms of DNA-methylation
mediated repression Klose and Bird, 2006
direct inhibition
of TF binding MBPs and
repressor
recruitment
DNMT/HDAC/HMT
linkage
Repression of
elongation
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38a
Nucleosome modification during transcription
Mellor, 2006
Rephrasing:
why cloned embryos are abnormal
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egg
sperm
zygote embryo+
Changes in methylation (Me) patterns (colors)
during development
Imprinted
Me patterns
Primordial
germ cellsnew Me
de-Me
H.Sive MIT 2006
new Me
Embryonic
Me patterns
adult
Cell type A
Cell type B
Cell type C
Cell type D
Cell type E
Cell type F
Adult Me patterns
new Me
Adult methylation patterns
do not normally
revert to embryonic patterns
Normal development
Methylation patterns (chromatin structure)
and correct gene expression
Egg/sperm
Embryo
Adult
“competent” state
early genes active
adult genes active
H.Sive MIT 2005
40
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DNA reprogramming
Adult donor nucleus
normal embryonic~0%
somewhat abnormal
may develop
Highly abnormal
failed
inject >> enucleated egg
After somatic nuclear transfer
Methylation patterns (chromatin) and
competence for correct gene expression
H.Sive MIT 2006
41
7. How to reprogram nuclei?
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adult donor nucleus
normal embryonic pattern>> pluripotent ES cells,
normal embryo
inject >> enucleated egg >> ICM
How to reprogram adult nucleus
to an embryonic state?
inject >> enucleated egg
Sequential
nuclear transfer
partially reprogrammed nucleus >> cell line
42
H.Sive MIT 2006
Serial nuclear transfer increases developmental potential
43
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adult donor nucleus/cell
normalembryonic nucleus/cell
Incubate in “reprogramming factors”
How to reprogram adult nucleus
to an embryonic state?44
H.Sive MIT 2006
Injection of mouse thymocyte nuclei into frog oocyte nucleus
Byrne et al, 2003
Oocyte nucleus
+ mouse nuclei
Low mag.
10 min post-inj. 5 days post-inj.
45
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Activation of oct4 expression in mouse thymocyte nuclei
injected into frog oocyte nuclei
Byrne et al, 2003
46
Simonsson and Gurdon, 2004
Deproteinization and demethylation decreases
delay in activation of oct4 expression
from mouse nuclei injected into frog oocytes47
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egg
sperm
zygote embryo+
Changes in methylation (Me) patterns (colors)
during development
Imprinted
Me patterns
Primordial
germ cellsnew Me
de-Me
H.Sive MIT 2006
new Me
Embryonic
Me patterns
adult
Cell type A
Cell type B
Cell type C
Cell type D
Cell type E
Cell type F
Adult Me patterns
new Me
ISSUE: different maternal and
paternal Me patterns
needed for normal development,
so may always need egg + sperm.
Nayernia et al, 2006
Functional sperm derived from ES-derived cells
Selection for
sperm markers
Embryos/pups
48
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Creating germ cells from ES cells (maybe)
Schöler et al 2003
Oct4 ZP3
49
8. Current and future issues
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Issues
EthicsUse of human embryos
Source of eggs/ surrogate mothers
Fate of abnormal babies
MethodsHow to clone primates?
ES cells >> eggs How to reprogram adult nuclei
Gene therapy
50