The role of the stemness factor Nanog in cell cycle regulation [Elektronische Ressource] / submitted by Bernhard Münst

Bernhard Münst

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2011
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

The
role
of
the
stemness
factor
Nanog 

in
cell
cycle
regulation





Thesis




Submitted
for
a
Doctoral
Degree
in
Natural
Sciences

(Dr.
rer.
nat.)



Faculty
of
Mathematics
and
Natural
Sciences 

Rheinische
Friedrich
Wilhelms
Universität
Bonn 







Submitted
by

Bernhard
Münst 





Bonn
2010 






Prepared
with
the
consent
of
the
Faculty
of
Mathematics
and
Natural
Sciences 

Rheinische
Friedrich
Wilhelms
Universität,
Bonn.



This
thesis
is
available
online
on
the
Hochschulschriftenserver
of
the
ULB
Bonn 

http://hss.ulb.u‐bnoinn.de/diss_online
.






Publication
year:
2011













































1.
Reviewer:
Prof.
Dr.
Oliver
Brüs
tle
2.
Reviewer:
Prof.
Dr.
Hubert
Scho
rle
Date
of
submission:
 15/09/2010 

Date
of
examination:
 22/12/2010 



I
Abbreviations 



AFP 
 
 
 
 alpha
feto‐protein 

AP
 
 
 
 alkaline
phosphatase

APS

 
 
 
 ammonium
persulfate

aa
 
 
 
 
 amino
acid 

BMP 
 
 
 
 bone
morphogenic
protein 

bp
 
 
 
 
 base
pair

BSA
 
 
 
 bovine
serum
albumine 

CDK
 
 
 
 cyclin
dependent
kinase 

cDNA 
 
 
 
 complementary
DNA

CKI
 
 
 
 cyclin
dependent
kinase
inhibitor 

cmc
 
 
 
 critical
micelle
concentration

D­MEM 

 
 
 Dulbecco's
Modified
Eagle
Medium 

DMSO

 
 
 
 dimethylsulfoxide 

DNA 
 
 
 
 deoxyribonucleic
acid

DTT
 
 
 
 dithiothreitol 

EB
 
 
 
 embryoid
body

EDTA

 
 
 
 ethylenediaminetetraacetic
acid 

EMSA
 
 
 
 electrophoretic
mobility
shift
assay 

EpiS
 
 
 
 epiblast
stem

ES
 
 
 
 embryonic
stem

FCS
 
 
 
 
 fetal
calf
serum

FGF
 
 
 
 fibroblast
growth
factor 

g
 
 
 
 
 gram

g
 
 
 
 
 gravitational
constant 

GAPDH 

 
 
 glyceraldehyde
3 ‐phosphate
dehydrogenase 

GSH 
 
 
 
 reduced
Gluthatione

GSK3
 
 
 
 glycogen
synthase
kinase
3 

GSSH 
 
 
 
 oxidized
Gluthatione

h

 
 
 
 hour

HEPES
 
 
 
 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic
acid 

HIV
 
 
 
 human
immunodeficiency
virus 

ICM
 
 
 
 inner
cell
mass 

IGF
 
 
 
 insulin‐like
growth
factor 

iPS
 
 
 
 induced
pluripotent
stem
cells

ITS
 
 
 
 Insulin
/
Transferrin
/
Selen ium

JAK
 
 
 
 Janus
Kinase

kb

 
 
 
 kilobase

kDa
 
 
 
 
 kiloDalton

II
KPC
 
 
 
 Kip1
ubiquitylation ‐promoting
complex

l

 
 
 
 liter

LB

 
 
 
 Luria
Bertani
Medium 

LIF

 
 
 
 leukemia
inhibitory
f actor

M
 
 
 
 
 Mol
per
liter

MALDI­TOF
 
 
 Matrix
Assisted
Laser
Desorption/Ionisation
 ‐
Time
Of
Flight 

MAPK
 
 
 
 mitogen‐activated
protein
kinase
 

MEF

 
 
 
 murine
embryonic
fibroblast 

MEK
 
 
 
 mitogen‐activated
protein
kinase
kinase 

MES
 
 
 
 2‐(N‐morpholino)ethanesulfonic
acid 

mg
 
 
 
 
 milligram

min

 
 
 
 minute

mL 
 
 
 
 millilit
er
mM
 
 
 
 
 millimol
ar
MWCO

 
 
 
 molecular
weight
cut‐off

NEAA
 
 
 
 non ‐essential
amino
acids 

ng

 
 
 
 nanogram

Ni­NTA
 
 
 
 Nickel‐nitrilotriacetic
acid

nm

 
 
 
 nanometer 

NLS

 
 
 
 nuclear
localization
signal

OD
 
 
 
 
 optical
density

PAGE
 
 
 
 polyacrylamid
electrophoresis 

PBS

 
 
 
 phosphate
buffe red
saline

PCR
 
 
 
 polymerase
chain
reaction 

PEG 
 
 
 
 polyethylene
glycol 

PI3K
 
 
 
 phosphatidylinositol
3 ‐kinase

PIPES
 
 
 
 piperazine‐N,N ′‐bis(2‐ethanesulfonic
acid) 

PP1 
 
 
 
 protein
phosphatase
type
1 

pRb 
 
 
 
 Retinoblastoma
protein

PrE
 
 
 
 primitive
endoderm

PTD
 
 
 
 protein
transduction
domain 

Rb 
 
 
 
 Retinoblastoma

RIS
 
 
 
 reprogramming
induces
senescence 

RNA 
 
 
 
 ribonucleic
acid

RT
 
 
 
 room
temperature
resp. 
reverse
transcription 

s
 
 
 
 second

SA
 
 
 
 senescence‐associated

SSEA
 
 
 
 stage‐specific
embryonic
antigen 

SDS

 
 
 
 sodium
dodecyl
sulfate

SMA 
 
 
 
 smooth
muscle
actin 

III
SMAD 
 
 
 
 small
mother
against
decapentaplegic

Stat3 
 
 
 
 signal
transducer
and
activator
of
transcription
3 

SV
40
 
 
 
 simian
virus
40 

TAT
 
 
 
 transactivator
of
transcription 

TB
 
 
 
 terrific
broth 

TBS
 
 
 
 Tris
buffe red
saline

TBS­T
 
 
 
 Tris
buffered 
saline
supplemented
with
Tween 

TE
 
 
 
 trophectoderm

TEMED

 
 
 tetramethylethylenediamine

TGFβ 
 
 
 
 transforming
growth
factor
beta 

Tris 
 
 
 
 tris(hydroxymethyl)aminomethane 


TUJ1 
 
 
 
 neuron ‐specific
class
III
bet‐atubulin

UV
 
 
 
 ultraviolet

V
 
 
 
 volt

rpm
 
 
 
 rounds
per
minute


IV Table of content
1
 Introduction .............................................................................................. 1

1.1 
 Embryonic
Stem
Cells.................... 1 

1.1.1
 Embryonic
development.........................1

1.1.2
 Derivation
and
characteristics
of
ES
cells........................4

1.1.3
 Derivatives
of
ES
cell................................s...............................5

1.2
 Extrinsic
signals
governing
pluripotency............................................... 6

1.2.1
 Murine
ES
cells............6 

1.2.1.1 
 LIF
signaling............................6 

1.2.1.2 
 BMP
signaling.........................................................7

1.2.2
 Human
ES
cells............................................7

1.2.2.1 
 Basic
FGF
signaling..............8

1.2.2.2 
 Activin
A
signaling................8

1.2.2.3 
 IGF
signaling9

1.2.3
 Epiblast
stem
cells......................................9

1.3
 Intrinsic
stemness
factors.......... 10 

1.3.1
 Oct3/4 ...........................................................................................10 

1.3.2
 Sox211

1.3.3
 Nanog.............................12

1.3.3.1 
 Expression
and
cellular
activities................................................12

1.3.3.2 
 Transcriptional
activities
and
protein
interaction...............12

1.3.3.3 
 Genetic
an alysis
of
Nanog
activity14

1.3.3.4 
 Analysis
of
stemness
factors
target
genes...............................15

1.4
 Cellular
reprogramming............................................. 16

1.5
 Cellular
senescence......................18

1.6
 Protein
transduction................................................... 18

1.7
 Aim
of
the
thesis............................22

2
 Materials
and
Methods ......................................... 23

2.1
 Materials.......................................... 23

2.1.1
 Chemicals.....................23

2.1.2
 Equipment...................................................23

2.1.3
 Enzymes .......................................................25

2.1.4
 Antibodies....................26 

2.1.5
 Buffers,
markers
and
medium............27

2.1.6
 Bacterial
strains........................................38

2.1.7
 Cell
lines38

2.1.8
 Expression
vectors...................................39

2.1.9
 Oligonucleotide
primers........................40 

V Table of content
2.2
 Nucleic
acids ................................................................... 41

2.2.1
 Generation
of
competent
 E.coli...........41

2.2.2
 Transformation
of
 E.coli........................41

2.2.3
 Preparation
of
small
amounts
of
DNA.............................41

2.2.4
 Preparation
of
large
amounts
of
DNA ................................42

2.2.5
 Photometric
measurement
of
DNA
content..................42

2.2.6
 Cloning
techniques..................................42

2.2.6.1 
 Restriction
hydrolysis.......42

2.2.6.2 
 Purification
of
DNA............42

2.2.6.3 
 Dephosporylation ...............................................43

2.2.6.4 
 Ligation...43

2.2.7
 Polymerase
chain
reaction...................................................43

2.2.8
 Reverse
transcriptase
polymerase
chain
reaction.....44

2.2.9
 Electrophoresis
in
agarose
gels..........45 

2.2.1
0 Retroviral
infection
and
iPS
induction.........................45 

2.3
 Proteins ............................................ 46

2.3.1
 Expression
of
recombinant 
fusion
proteins..................................................46 

2.3.2
 Imidazole
gradient...46 

2.3.3
 Purification
of
recombinant
fusion
proteins................47 

2.3.3.1 
 Denaturing
purification ...................................47

2.3.3.2 
 Native
purification.............47

2.3.4
 Sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis48

2.3.5
 Immunoblot................49

2.3.6
 Electrophoretic
Mobility
Shift
Assay................................50 

2.3.7
 Immunocytochemistry...........................51

2.4
 Cell
culture ...................................... 52

2.4.1
 General
methods.......52

2.4.1.1 
 Passaging
of
cells................52

2.4. 1.2
 Counting
cells.......................................................52

2.4.1.3 
 Freezing
and
thawing
cells.............................53

2.4.1.4 
 Embryoid
body
formation..............................................................53

2.4.2
 Protein
transduction...............................53

2.4.2.1 
 Oct3/4
GiP
MEFs
and
CV1
fibroblast
cells53

2.4.2.2 
 MP‐AF
primary
hu