12R-lipoxygenase deficient mouse skin models for autosomal recessive congenital ichthyosis [Elektronische Ressource] / presented by Silvia de Juanes

De

Dissertation


Submitted
to
 the
Combined
Faculties
for
the
Natural
Sciences
and
for
Mathematics 
of
the
Ruperto-Carola
University
of
Heidelberg,
Germany

for
the
degree
of 
Doctor
of
Natural
Sciences




















presented
by 
Silvia
de
Juanes
born
in:
Madrid,
Spain

Oral
examination:
29.04.
09




12R-LIPOXYGENASE
DEFICIENT
 
MOUSE
 SKIN
MODELS
 
FOR
AUTOSOMAL
RECESSIVE
CONGENITAL
ICHTHYOSIS 






















Referees:
Prof.
Dr.
Rainer
Zawa
tskyPD
Dr.
Karin
Mülle-rDecker































To A lex Part
of
this
work
was
published
in: 
de
Juanes
S,
Epp
N,
Latzko
S,
Neumann
M,
Fürsteberger
G,
Hausser
I,
Stark
HJ
and
Krieg
P.
Development
of
an
Ichthyosiform
Phenotype
in
Alox12b -Deficient
Mouse
Skin
Transplants.
 JID
(2009).

de
Juanes
S,
Epp
N,
Fürsteberger
G,
Müller
K,
Leitges
M,
Hausser
I,
Thieme
F,
Liebisch
G,
Schnitz
G,
Stark
HJ
and
Krieg
P.
Knockout
mouse
models
for
the
exploration
of
the
role
of
epidermal
Lipoxygenases
in
epidermal
barrier
formation
and
terminal
differentiation
in
skin.

Leopoldina
symposium
on
Lipid
Signalling
(2008)
Book
of
abstracts.
 
Krieg
P,
Epp
N,
Fürsteberger
G,
de
Juanes
S ,
Eckl
KM,
Hennies
HC,
Hausser
I,
Thieme
F,
Liebisch
G,
Schnitz
G
and
Stark
HJ.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 28
Tags :
Source : ARCHIV.UB.UNI-HEIDELBERG.DE/VOLLTEXTSERVER/VOLLTEXTE/2009/9425/PDF/SILVIA_DE_JUANES.PDF
Nombre de pages : 106
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Dissertation



Submitted
to
 the

Combined
Faculties
for
the
Natural
Sciences
and
for
Mathematics 

of
the
Ruperto-Carola
University
of
Heidelberg,
Germany


for
the
degree
of 

Doctor
of
Natural
Sciences





















presented
by 

Silvia
de
Juanes

born
in:
Madrid,
Spain


Oral
examination:
29.04.
09





12R-LIPOXYGENASE
DEFICIENT
 

MOUSE
 SKIN
MODELS
 

FOR
AUTOSOMAL
RECESSIVE
CONGENITAL

ICHTHYOSIS 























Referees:

Prof.
Dr.
Rainer
Zawa
tsky
PD
Dr.
Karin
Mülle-rDecker
































To A lex Part
of
this
work
was
published
in: 

de
Juanes
S,
Epp
N,
Latzko
S,
Neumann
M,
Fürsteberger
G,
Hausser
I,

Stark
HJ
and
Krieg
P.
Development
of
an
Ichthyosiform
Phenotype
in

Alox12b -Deficient
Mouse
Skin
Transplants.
 JID
(2009).


de
Juanes
S,
Epp
N,
Fürsteberger
G,
Müller
K,
Leitges
M,
Hausser
I,

Thieme
F,
Liebisch
G,
Schnitz
G,
Stark
HJ
and
Krieg
P.
Knockout
mouse

models
for
the
exploration
of
the
role
of
epidermal
Lipoxygenases
in

epidermal
barrier
formation
and
terminal
differentiation
in
skin.

Leopoldina

symposium
on
Lipid
Signalling
(2008)
Book
of
abstracts.
 

Krieg
P,
Epp
N,
Fürsteberger
G,
de
Juanes
S ,
Eckl
KM,
Hennies
HC,

Hausser
I,
Thieme
F,
Liebisch
G,
Schnitz
G
and
Stark
HJ.
A
novel
eicosaneid

pathway
is
essential
for
the
epidermal
barrier
function:
lessons 
from

congenital
ichthyosis
and
12R -lipoxygenase
deficient
mice.

Leopoldina

symposium
on
lipid
signalling
(2008)
Book
of
abstracts.
 

Epp
N,
Fürsteberger
G,
Müller
K,
de
Juanes
S ,
Leitges
M,
Hausser
I,

Thieme
F,
Liebisch
G,
Schnitz
G
and
Krieg
P.
12R-lipox ygenase
deficiency

disrupts
epidermal
barrier
function.
JCB
(2007)
 179(4):747-60. 
Epp
N,
de
Juanes
S ,
Fürsteberger
G,
Müller
K,
Leitges
M,
Hausser
I,

Thieme
F,
Liebisch
G,
Schnitz
G,
Stark
HJ
and
Krieg
P.
12R-lipoxygenase

deficiency
disrupts
epidermal
barr ier
function.
First
World
Conference
on

Ichthyosis
(2007)
Book
of
abstracts.

de
Juanes
S,
Epp
N,
Fürsteberger
G,
Müller
K,
Leitges
M,
Hausser
I,

Thieme
F,
Liebisch
G,
Schnitz
G,
Stark
HJ
and
Krieg
P.
12R-Lipoxygenase
is

essential
for
the
epidermal
barrier
 function:
lessons
from
12R -lipoxygenase

thdeficient
mice.
37 
Annual
ESDR
meeting
(2007)
Book
of
abstracts. 

Eckl
KM,
Torres
S,
 de
Juanes
S,
Metze
D,
Krieg
P
and
Hennies
HC.

Functional
model
systems
for
congenital
ichthyosis:
Basic
and
long
way
to

ththerapy.
37 



Other
publications: 

Eckl
KM
,
 de
Juanes
S,
Kurtenbach
J,
Natebus
M,
Lugassy
J,
Oji
V,
Traupe

H,
Preil
ML,
Martínez
F,
Smolle
J,
Harel
A,
Krieg
P,
Sprecher
E
and
Hennies

HC.
Molecular
Analysis
of
250
Pati ents
with
Autosomal
Recessive
Congenital

Ichthyosis:
Evidence
for
Mutation
Hotspots
in
 ALOXE3
and
Allelic

Heterogeneity
in
 ALOX12B.
JID
(2009)
Table of contents 
Table
of
contents 

Abbreviations
…………………………………………………………
. 9

Summary
……………………………………………………………….. 
 11

Zusammenfassung
…………………………………………………… 
 13

1.
Introduction
………………………………………………………
…15

1.1.
The
Skin
…………………………………………………………. 
 15

1.1.1.
Epidermis:
structure
and
function
 …………………………. 
 15

1.1.2.
Terminal
differentiation
of
the
epidermis
 ………………….. 
 16

1.2.
Ichthyosis
………………………………………………………… 
 17

1.3.
Lipoxygenases
…………………………………………………… 
 18

1.3.1.
Structure
…………………………………………………… 
 19

1.3.2.
Function
……………………………………………………. 
 2 0 

1.3.3.
Classification
………………………………………………. 
 2 0 

1.3.4.
Epidermal
lipoxygenases
 …………………………………. 
 2 1

1.3.5.
12R -LOX
and
 eLOX-3
…………………………………….. 
 2 2 

1.4.
Genetically
modified
animals
…………………………………….. 
 2 3

1.4.1.
Conditional
Knockout
 ……………………………………… 
 2 3

1.4.1.1.
Inducible
conditional
Knockout 
…………………….. 
 2 3

1.5.
12R-LOX
deficient
mouse
…………………………………………… 
 2 4

1.6.
Aim
of
the
project
 ………………………………………………… 
 2 6

2.
Materials…………………………………………………………….. 
 2 7

2.1.
Chemicals
 ………………………………………………………… 
 2 7

2.2.
Kits
 ……………………………………………………………….. 
 29

2.3.
Antobodies
……………………………………………………….. 
 30 

2.3.1.
Primary
antibodies
…………………………………………. 
 30 

2.3.2.
Secondary
antibodies
 ……………………………………… 
 32 

  5 Table of contents 
2.4.
Equipments
and
devices
 …………………………………………. 
 32 

2.5.
Oligonucleotids
…………………………………………………… 
 35

2.6.
DNA
and
Protein
Ladders
……………………………………….. 
 36

2.7.
Mouse
strains
 …………………………………………………….. 
 36

2.8.
Computer
Software
 ………………………………………………. 
 36

3.
Methods
……………………………………………………………..
 38

3.1.
DNA
analysis
...…………………………………………………… 
 38

3.1.1.
Extraction
of
genomic
DNA
from
tail
biopsy
………………… 
 38

3.1.2.
DNA
concentration
 ………………………………………… 
 38

3.1.3.
Amplification
of
DNA
 ………………………………………. 
 38

3.1.4.
Agarose
gel
electrophoresis
 …………………………….. 
 39

3.2.
Preparation
of
epidermis
extracts
 ……………………………….. 
 40 

3.3.
RNA
analysis
…………………………………………………….. 
 40 

3.4.
Protein
analysis
………………………………………………….. 
 40 

3.4.1.
Protein
extraction
 ………………………………………….. 
 40 

3.4.2.
DC
quick
Lowry
for
measuring
protein
concentrations
……. 
 40 

3.4.3.
Discontinuous
SDS-polyacrilamide
gel
electrophoresis
…… 
 41

3.4.4.
Immunoblot
analysis
 –
western
transfer
…………………….. 
 41

3.4.5.
Immunoblot
analysis -
incubation
with
antibodies
…………...
 
 42

3.5
Preparation
and
staining
of
skin
sections
…………………………... 
 43

3.5.1.
Preparation
of
cryo -sections
………………………………….. 
 43

3.5.2.
Immunohistochemical
staining
……………………………….. 
 43

3.5.3.
Preparation
of
paraffin
sections
……………………………… 
 43

3.5.4.
Hematoxylin
and
Eosin
staining
……………………………… 
 43

3.5.5.
Electron
microscopy
 ……………………………………….. 
 44

3.6.
Animal
work
 ………………………………………………………. 
 44

3.6.1.
Mouse
maintenance
…………………………………………… 
 44

  6 Table of contents 
3.6.2.
Transepidermal
water
loss
measurement
…………………... 
 44

3.6.3.
Epidermis
isolation
from
newborn
mice
……………………... 
 44

3.6.4.
Epidermis
isolation
from
adult
mice
…………………………. 
 45

3.6.5.
Skin
grafting
 ……………………………………………….. 
 45

3.6.6.
Tamoxifen
treatment
 ………………………………………. 
 45

4.
Results
………………………………………………………………
. 46

-/-4.1.
Characterization
of
12R -LOX 
adult
skin
phenotype
...…………. 
 46

-/-
4.1.1.
Genotyping
strategy
for
detection
of
12R-LOX neonates
..
 46

4.1.2.
Macroscopic
characterisation
of
12R -LOX
skin
grafts
……. 
 46

4.1.3.
Ultrastructural
analysis
……………………………………… ...
 47

4.1.4.
Hyperproliferation
in
skin
grafted
epidermis
………………… 
 49

4.1.5.
Comparative
analysis
of
differentiation
markers
………… …
 50 

4.1.6.
Transepidermal
water
loss
 …………………………………… 
 53

4.1.7.
Summary

 ………………………………………………….. 
 54

4.2.
Conditional
inactivation
of
12R -LOX
in
mouse
skin
………………. 
 55

4.2.1.
Strategy
to
generate
temporally
controlled
inactivation
of
 



12R-LOX
in
mouse
epidermis
……………………………………….. 
 55

fl/fl T24.2.2.
Genotyping
of
 Alox12b 
and
K14-CreER 
via
PCR
of
 



genomic
DNA
 …………………………………………………….. 
 57

fl/fl/K1-C4re -ERT2
4.2.3.
Development
of
mutant
12R -LOX mice
………. 
 58

4.2.3.1
Body
Weight
 ………………………………………. 
 58

4.2.3.2.
Morphological
examination
…………………………. 
 59

4.2.3.3
Skin
histology
 ……………………………………… 
 62

4.2.3.4
Molecular
analysis
of
spontaneous
activation
of
the
 



Cre
recombinase
 ………………………………………….. 
 64

4.2.3.4.1.
Spontaneous
Cre -mediated
inactivation
of
 



12R-LOX
in
the
epidermis
of
mutant
mice
…………….. 
 64

4.2.3.4.2.
Spontaneous
loss
of
12R -LOX
protein
 




expression
in
mutant
mice
epidermis
…………………... 
 65

  7 Table of contents 
4.2.3.
Summary
 …………………………………………………… 
 66

flox/flox4.3.
Tamoxifen
induced
deletion
of
12R -LOX
in
Alox12b 
K14- 



T2Cre -ER 
mice
 ………………………………………………………… 
 67

4.3.1
Morphological
examination
 ………………………………… 
 68

4.3.2
Histological
analysis
 ……………………………………….. 
 70 

4.3.3.
Cre -mediated
deletion
of
Alox12b 
is
restricted
to
Keratin
 



14 -expressing
tissues
……………………………………………. 
 71

4.3.4.
Epidermal
differentiation
and
proliferation
markers
 ………..
 72 

4.3.5.
Summary
 …………………………………………………… 
 74

4.4.
Insights
in
the
12R -LOX
molecular
pathway
………………………. 
 75

4.4.1.
Gene
expression
profile
of
12R -LOX
deficient
mice
………. 
 75

4.4.2.
Up -regulation
of
AP -1
complex
proteins
…………………….. 
 77

4.4.3.
Summary
 …………………………………………………… 
 79

5.
Discussion…………………………………………………………..
 
 80 

5.1.
Animal
models 
generated
to
study 
12R-LOX
deficiency
in
the
 81

adult
stage
…………………………………………………… ……….. 

fl/fl/K1-C4re -ERT25.2.
Ichthyosiform
phenotype
in
skin
grafts
and
 Alox12b 
 82 

mouse
 …………………………………………………… ……………. 

5.3.
Ultrastructure
of
the
12R -LOX
skin
……………………………… 
 84

5.4.
Other
phenotypic
changes
observed
in
the
12R -LOX
conditional
 



mouse
model 
…………………………………………………… …….. 
 84

5.5.
Cornified
envelope
proteins 
……………………………………… 
 85

5.6.
Involvement
of
12R -LOX
in
skin
differentiation
pathways
 ……….. 
 86

Supplementary
table
. …………………………………………….. 
 90 

References
…………………………………………………………….. 
 97

Figures
…………………………………………………………………. 
 104

Acknowledgements
…………………………………………………..
 106

  8 Abbreviations
Abbreviations

4-OHT 
 4-hydroxy-tamoxifen 
AP-1
 Activating
Protei-1n

APS
 Ammonium
persulfate 

ARCI
 Autosomal
 Recesiv
Congenital
Ichthyosis

BSA
 Bovine
serum
albumin 

CE 
 Cornified
Envelope 

CE 
 Cornified
envelope  
CLE 
 Cornified
Lipid
Envelop
e
CLE 
 Cornified
lipid
envelop e
Cre 
 Causes
Recombination  
Da 
 Dalton

del
 Delete 

DKFZ
 Deutsches
Krebsforchungszentrum 

DNA
 Deoxyribonucleic
acid 

DNase 
 Deoxyribonuclease 

dNTP
 Deoxynocleosidetriphosphate 

ER
 Estrogen
Receptor  
Erk
 Extracellular -signalling
regulated
kinase  
EtOH 
 Ethanol

Fig
 Figure

Fi
l Filaggrin 
fl
 Floxed 
FLP
 FliPase
Recombianse
  
FRT 
 Flp
Recognition
Target  
GAPDH 
 Glycerol -aldehyd-3-phosphat -dehydrogenase

h
 Hour 

HETE 
 Hydroxyeicosatetraionic
acid

HODE 
 hydroxyoctadecadienoic
acids

HPETE 
 Hydroxyperoxyeicosatetraionic
acid 

In
v Involucrin 
3K
 Kilo,
10

kb
 Kilobasepair 

KO 
 Knockout 
LB
 Lamellar
Bodies 

LBD
 Ligand
Binding
Dom ain
  9 Abbreviations
LI
 Lamel lar
Ichthyosis

LOX
 Lipoxygenase

LoxP
 Location
of
crossing-over
in
bacteriophage
P1 

M 
 Molar=
mol/l 

m 
 Meter 

mAb 
 Monoclonal
an tibody

mg 
 Milligram

min
 Minut
e
ml
 
 Millilit
re
mRNA 
 Messenger
ribonucleic
acid 

NCIE 
 Non-bullous
Congenital
Ichthyosiform
Erythroderma 

n/u
 Not
used

o/n
 Overnight

OD 
 Optic
densit
y
pAb
 Polyclonal
antibod
y
PAGE
 Polyacrilamide
gel
electrophoresis 

PBS
 Phosphate
buffered
saline

PBS-T 
 Phosphate
buffered
saline
–
Tween
20 

PCR 
 Polymerase
 Chain
Reaction

PFA
 Paraformaldehyde 

RNA
 Ribonucleic
acid

RNase 
 Ribonuclease 

ROS 
 Reactive
Oxygen
Species 

rpm 
 Revolutions
per
minute

Rptn
 Repetin 
RT 
 Room
temperature 

RT -PCR 
 Reverse
transcriptase -
polymerase
chain
reaction 

SDS
 Sodium
dodecyl
sulfate

SPRR
 Small
PRoline
Rich 
Tam 
 Tamoxifen 

TEMED 
 N,N,N’,-NTe’tramethylethylenediamine

TEWL 
 Tra nsepidermal
Water
Loss 

TGM1 
 transglutaminase
1

Tm 
 Melting
temperature 

wt
 Wild-type 
-6µ
 Micro,
10 

  10 

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