RNA regulons during heart development and regeneration:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
Köln
[2020]
|
| Subjects: | |
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| 100 | 1 | |a Yao, Wenjie |e Verfasser |0 (DE-588)1217441034 |4 aut | |
| 245 | 1 | 0 | |a RNA regulons during heart development and regeneration |c Wenjie Yao |
| 264 | 1 | |a Köln |c [2020] | |
| 300 | |a 174, 1-2 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität zu Köln |d 2020 | ||
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Record in the Search Index
| _version_ | 1804183385570017280 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
1
ABSTRACT
----------------------------------------------------------------------------------------------------------------------------1
2
INTRODUCTION
---------------------------------------------------------------------------------------------------------------------
6
2.1
PROGRAMMING
CELL
FATES
-------------------------------------------------------------------------------------------------
7
2.1.1
CELL
FATE
REGULATION
IN
EARLY
MAMMALIAN
DEVELOPMENT
--------------------------------------------------------------------
7
2.1.2
CARDIOMYOCYTES,
THE
FUNCTIONAL
UNITS
OF
THE
HEART
-------------------------------------------------------------------------
11
2.1.3
MODELING
CARDIAC
LINEAGE
COMMITMENT
IN
DEFINED
CONDITIONS
USING
HPSCS
-------------------------------------
13
2.2
REPROGRAMMING
CELL
FATES
-------------------------------------------------------------------------------------------
14
2.2.1
CELL
FATE
CONVERSION
-----------------------------------------------------------------------------------------------------------------------
14
2.2.2
CELL
FATE
REPROGRAMMING
DURING
HEART
REGENERATION
-----------------------------------------------------------------------
16
2.2.3
DEVELOPMENT
VERSUS
REGENERATION
OF
THE
HEART
------------------------------------------------------------------------------
17
2.3
MODELS
USED
IN
THE
THESIS
FOR
STUDYING
CELL
FATE
DECISIONS
---------------------------------------------
18
2.3.1
IN
VITRO
HESC-BASED
MODEL
FOR
HEART
DEVELOPMENT
------------------------------------------------------------------------
18
2.3.2
HEART
REGENERATION
MODEL
IN
ZEBRAFISH
---------------------------------------
20
2.4
MOLECULAR
REGULATIONS
OF
CELL
FATE
DECISIONS
------------------------------------------------------------------
21
2.4.1
SIGNALING
PATHWAYS
REGULATING
CELL
FATE
DECISIONS
IN
THE
CONTEXT
OF
THE
HEART
--------------------------------
23
2.4.2
EPIGENETIC
MACHINERY
REGULATING
CELL
FATE
DECISIONS
-----------------------------------------------------------------------
27
2.4.3
TRANSCRIPTION
FACTORS
REGULATE
CELL
FATE
DECISIONS
---------------------------------------------------------------------------
29
2.4.4
CO
AND
POST-TRANSCRIPTIONAL
REGULATORS
IN
CELL
FATE
DECISIONS
---------------------------------------------------------
32
3
AIMS
OF
THIS
THESIS
----------------------------------------------------------------------------------------------------------
41
4
RESULTS
----------------------------------------------------------------
---------------------------------------------------------
43
4.1
PART
I
------------------------------------------------------------------------------------------------------------------------
43
4.1.1
QKI
IS
CONSERVED
IN
THE
VERTEBRATES
----------------------------------------------------------------------------------------------
43
4.1.2
DEVELOPMENTAL
EXPRESSION
PROFILES
OF
QKI
IN
HUMAN
IN
VITRO
CARDIOMYOCYTE
DIFFERENTIATION
MODEL
-45
4.1.3
SUBCELLULAR
LOCALIZATION
OF
QKI
------------------------------------------------------------------------------------------------------
47
4.1.4
CRISPR/CAS9-MEDIATED
GENERATION
OF
QKI
KNOCKOUT
HESC
LINES
------------------------------------------------------
50
4.1.5
VALIDATION
OF
PLURIPOTENCY
UPON
DEPLETION
OF
QKI
IN
HESCS
-------------------------------------------------------------
52
4.1.6
DECIPHERING
THE
ROLE
OF
QKI
IN
LINEAGE
COMMITMENT
OF
HESCS
--------------------------------------------------------
54
4.1.7
MECHANISTIC
INSIGHTS
INTO
THE
FUNCTION
OF
QKI
DURING
CARDIAC
DIFFERENTIATION
---------------
:
-----------------
66
4.2
PART
II
------------------------------------------------------------------------------------------------
-
-----------------
73
4.2.1
GLOBAL
TRANSCRIPTOMIC
ANALYSIS
DURING
ZEBRAFISH
HEART
REGENERATION
----------------------------------------------
73
4.2.2
IDENTIFICATION
OF
MIRNA-DEPENDENT
GENE
REGULATORY
NETWORK
CONTROLLING
ZEBRAFISH
HEART
REGENERATION
---------------------------------------------------------------------------------------------------------------------------------------------
78
4.2.3
FUNCTIONAL
CHARACTERIZATION
OF
DRE-MIR-429A
DURING
ZEBRAFISH
HEART
REGENERATION
-------------------------
83
5
DISCUSSIONS
-
-
-
-
-
-
-
--
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
88
5.1
PART
I------------------------------------------------------------------------------------------------------------------------88
5.1.1
QKI
IS
AN
EVOLUTIONARIIY
CONSERVED
RBP
ENRICHED
IN
THE
NUCLEUS
DURING
THE
CARDIAC
COMMITMENT
OF
HUMAN
EMBRYONIC
STEM
CELLS
-----------------------------------------------------------------------------------------------------------------
88
5.1.2
QKI
IS
NOT
ESSENTIAL
FOR
THE
INDUCTION
OF
GERM
LAYERS
----------------------------------------------------------------------
89
5.1.3
THE
INTERPLAY
OF
QKI
WITH
WNT
SIGNALING
IS
CRITICAL
FOR
CARDIAC
MESODERM
SPECIFICATION
-------------------
90
5.1.4
QKI
LIKELY
SETS
THE
MRNA
SPLICING
CODE
FOR
CARDIAC
DIFFERENTIATION
AND
CONTRACTILITY
-------------------------91
5.1.5
QKI
DIRECTLY
TARGETS
THE
PRE-MRNA
OF
GENES
INVOLVED
IN
WNT
SIGNALING
AND
CARDIAC
MYOFIBRIL
FORMATION
--------------------------------------------------------------------------------------------------------------------------------------------------
95
5.1.6
CONCLUSION
I
---------------------------------------------------------------------------------------------------------------------------------
97
5.1.7
OUTLOOKS
I
-------------------------------------------------------------------------------------------------------------------------------------98
5.2
PART
II
----------------------------------------------------------------------------------------------------------------------
99
5.2.1
COMBINED
MIRNA AND
MRNA
PROFILING
REVEALS
THAT
HEART
REGENERATION
IS
A
TRANSIENT
CELLULAR
REPROGRAMMING
PROCESS
----------------------------------------------------------------------------------------------------------------------------
99
5.2.2
IDENTIFICATION
AND
RANKING
OF
MIRNA-DEPENDENT
GENE
REGULATORY
NETWORKS
DURING
ZEBRAFISH
HEART
REGENERATION
----------------------------------------------------------------------------------------------------------------------------------
101
5.2.3
THE
DRE-MIR-429A
IS
A
CRUCIAL
REGULATOR
FOR
CM
PROLIFERATION
DURING
ZEBRAFISH
CARDIAC
REGENERATION
--------------------------------------------------------------------------------------------------------------------------------------------
102
5.2.4
CONCLUSION
II
------------------------------------------------------------------------------------------------------------------------------104
5.2.5
OUTLOOKS
II
---------------------------------------------------------------------------------------------------------------------------------
104
6
MATERIAL
AND
METHODS
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
105
6.1
MATERIALS
---------------------------------------------------------------------------------------------------------------
105
6.1.1
CHEMICALS
----------------------------------------------------------------------------------------------------------------------------------
105
6.1.2
ANTIBODIES
......................................
107
6.1.3
PLASMIDS--------------------------------------------------------------------------------------------------------------------------
*
--------
109
6.1.4
BACTERIAL
STRAINS
---------------------------------------------------------------------------------------------
110
6.1.5
HUMAN
CELL
LINES
....................
-
-
-
---------------------
110
6.1.6
MICE
CELL
LINES
----------------------------------------------------------------------------------------------------------------------------110
6.1.7
ZEBRAFISH
LINES
---------------------------------------------------------------------------------------------------------------------------110
6.2
ANIMAL
HUSBANDRY
AND
ANIMAL
EXPERIMENT
----------------------------------------------------------------
111
6.2.1
ZEBRAFISH
HUSBANDRY
------------------------------------------------------------------------------------------------------------------
HL
6.2.2
CARDIAC
INJURY
------------------------------------------------------------
-
--------------
-
------------------------------------
111
6.2.3
INTRA-CARDIAC
INJECTION
OF
MIRNA
INHIBITOR
OR
MO
-----------------------------------------------------------------------
111
6.2.4
ZEBRAFISH
BREEDING
AND
MICROINJECTION
-
-
---------------------------------------------------------------
HL
6.2.5
COLLECTION
OF
THE
HEARTS
POST-INJURY
--------------------------------------------------------------------------------------------
112
6.2.6
PREPARATION
OF
THE
HISTOLOGICAL
SAMPLES
-------------------------------------------------------------------------------------
113
6.3
CELL
CULTURE
-------------------------------------------------------------------------------------------------------------
113
6.3.1
COATING
OF
CELL
CULTURE
DISHES
WITH
MATRIGEL
----------------------------------------------------------------------------------
113
6.3.2
MAINTENANCE
CULTURE
OF
HESCS
-----------------------------------------------------------------------------------------------------
113
6.3.3
FREEZING
OF
HESCS
FOR
LONG-TERM
STORAGE
--------------------------------------------------------------------------------------
114
6.3.4
CARDIOMYOCYTE
DIFFERENTIATION
-----------------------------------------------------------------------------------------------------
114
6.3.5
ADDITIONAL
MESODERM
DIFFERENTIATION
PROTOCOLS
---------------------------------------------------------------------------
115
6.3.6
INDUCTION
OF
ECTODERM
AND
ENDODERM
LINEAGE
DIFFERENTIATION
-----------------------------------------------------
116
6.3.7
GENERATION
OF
CRISPR/CAS9-MEDIATED
KNOCKOUT
OF
GENES
IN
HESCS-----------------------------------------------
116
6.3.8
GENERATION
OF
STABLE
DOXYCYCLINE-INDUCIBLE
QKI
OVEREXPRESSION
LINES
IN
QKI-KO
HESCS
-----------------
117
6.4
MOLECULAR
BIOLOGY
----------------------------------------------------------------------------------
118
6.4.1
BACTERIA
GROWTH
CONDITIONS
---------------------------------------------------------------------------------------------------------
118
6.4.2
TRANSFORMATION
OF
BACTERIA
---------------------------------------------------------------------------------------------------------
118
6.4.3
PLASMID
ISOLATION
-------------------------------------------------------------------------------------------------------------------------
119
6.4.4
PCR
PRIMERS
--------------------------------------------------------------------------------------------------------------------------------
119
6.4.5
POLYMERASE
CHAIN
REACTION
(PCR)
-------------------------------------------------------------------------------------------------
120
6.4.6
COLONY
PCR
---------------------------------------------------------------------------------------------------------------------------------
121
6.4.7
AGAROSE
GEL
ELECTROPHORESIS
--------------------------------------------------------------------------------------------------------
122
6.4.8
GENERATION
OF
CRISPR
PLASMIDS
----------------------------------------------------------------------------------------------------
122
6.4.9
SGRNA
TEMPLATE
ASSEMBLY
AND
PREPARATION
OF
SGRNA
-------------------------------------------------------------------
123
6.4.10
RNA
EXTRACTION
-----------------------------------------------------------------------------------------------------------------------
124
6.4.11
DNASE
TREATMENT
OF
PURIFIED
RNA
--------------------------------------------------------------------------------------------
124
6.4.12
REVERSE
TRANSCRIPTION
--------------------------------------------------------------------------------------------------------------
124
6.4.13
RNA
PRECIPITATION
-------------------------------------------------------------------------------------------------------------------
125
6.4.14
SEMI-QUANTITATIVE
REAL-TIME
PCR
(QPCR)
---------------------------------------------------------------------------------
126
6.4.15
MIRNA
QPCR
--------------------------------------------------------------------------------------------------------------------------
127
6.4.16
ENHANCED
CROSS-LINKING
IMMUNOPRECIPITATION
FOLLOWED
BY SEQUENCING
(ECLIP-SEQ)
-------------------
128
6.5
PROTEIN
BIOCHEMISTRY
----------------------------------------------------------------------------------------------
128
6.5.1
PROTEIN
ISOLATION
-------------------------------------------------------------------------------------------------------------------------
128
6.5.2
SODIUM
DODECYL
SULFATE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
--------------------------------------
129
6.5.3
WET
BLOTTING
OF
PROTEINS
ON
POLYVINYLIDENE
DIFLUORIDE
(PVDF)
MEMBRANES
-----------------------------------
130
6.5.4
IMMUNOPRECIPITATION
FOR
MASS
SPECTROMETRY
AND
WB
-----------------------------------------------------------------
130
6.5.5
NUCLEAR-CYTOSOLIC
FRACTIONATION
........
131
6.6
HISTOLOGICAL
EXPERIMENT
-------------------------------------------------------------
-
---------------------------
131
6.6.1
SURIS
RED
AND
FAST
GREEN
STAINING
---------------------------
131
6.6.2
IMMUNOCYTOCHEMISTRY
----------------------------------------------------------------------------------------------------------------
132
6.6.3
MITO-TRACKER
STAINING
------------------------------------------------------------------------------------------------------------------
132
6.6.4
ER
TRACKER
STAINING
---------------------------------------------------------------------------------------------------------------------
133
6.7
RTCA
TO
MEASURE
BEATING
ACTIVITY
OF
HESC-CMS
-------------------------------------------------------
133
6.8
BIOINFORMATIC
ANALYSIS
-------------------------------------------------------------------------------------------
134
6.8.1
RNA
SEQUENCING
DATA
ANALYSIS
-------
--------------------------------------------------------------------------------------------
134
6.8.2
PREDICTION
OF
MIRNA
TARGETS
-------------------------------------------------------------------------------------------------------
134
6.8.3
IDENTIFICATION
OF
MIRNA-DEPENDENT
NETWORKS
REGULATING
HEART
REGENERATION
--------------------------------
134
6.8.4
PROTEOMIC
DATA
ANALYSIS
-------------------------------------------------------------------------------------------------------------
134
6.8.5
ECLIP
ANALYSIS
-----------------------------------------------------------------------------------------------------------------------------
134
6.9
DATA
ANALYSIS
AND
PLOTTING
-------------------------------------------------------------------------------------135
6.9.1
STATISTICAL
ANALYSIS
----------------------------------------------------------------------------------------------------------------------
135
6.9.2
CORRELATION
ANALYSIS
--------------------------------------------------------------------------------------------------------------------
135
6.9.3
PRINCIPAL
COMPONENT
ANALYSIS
(PCA)
--------------------------------------------------------------------------------------------
135
6.9.4
HEATMAPS
--------------------------------------------------------------------------------------------------------------------------------
~
135
6.9.5
CIRCLE
PLOTS
----------------------------------------------------------------------------------------------------------------------------------
135
6.9.6
GO
ENRICHMENT
ANALYSIS
-------------------------------------------------------------------------------------------------------------
135
7
REFERENCES
------------------------------------------------
136
8
ACKNOWLEDGEMENTS
-
172
9
ERKLARUNG
ZUR
DISSERTATION
-------------------------------------------------------------------------------------------
174
|
| adam_txt |
TABLE
OF
CONTENTS
1
ABSTRACT
----------------------------------------------------------------------------------------------------------------------------1
2
INTRODUCTION
---------------------------------------------------------------------------------------------------------------------
6
2.1
PROGRAMMING
CELL
FATES
-------------------------------------------------------------------------------------------------
7
2.1.1
CELL
FATE
REGULATION
IN
EARLY
MAMMALIAN
DEVELOPMENT
--------------------------------------------------------------------
7
2.1.2
CARDIOMYOCYTES,
THE
FUNCTIONAL
UNITS
OF
THE
HEART
-------------------------------------------------------------------------
11
2.1.3
MODELING
CARDIAC
LINEAGE
COMMITMENT
IN
DEFINED
CONDITIONS
USING
HPSCS
-------------------------------------
13
2.2
REPROGRAMMING
CELL
FATES
-------------------------------------------------------------------------------------------
14
2.2.1
CELL
FATE
CONVERSION
-----------------------------------------------------------------------------------------------------------------------
14
2.2.2
CELL
FATE
REPROGRAMMING
DURING
HEART
REGENERATION
-----------------------------------------------------------------------
16
2.2.3
DEVELOPMENT
VERSUS
REGENERATION
OF
THE
HEART
------------------------------------------------------------------------------
17
2.3
MODELS
USED
IN
THE
THESIS
FOR
STUDYING
CELL
FATE
DECISIONS
---------------------------------------------
18
2.3.1
IN
VITRO
HESC-BASED
MODEL
FOR
HEART
DEVELOPMENT
------------------------------------------------------------------------
18
2.3.2
HEART
REGENERATION
MODEL
IN
ZEBRAFISH
---------------------------------------
20
2.4
MOLECULAR
REGULATIONS
OF
CELL
FATE
DECISIONS
------------------------------------------------------------------
21
2.4.1
SIGNALING
PATHWAYS
REGULATING
CELL
FATE
DECISIONS
IN
THE
CONTEXT
OF
THE
HEART
--------------------------------
23
2.4.2
EPIGENETIC
MACHINERY
REGULATING
CELL
FATE
DECISIONS
-----------------------------------------------------------------------
27
2.4.3
TRANSCRIPTION
FACTORS
REGULATE
CELL
FATE
DECISIONS
---------------------------------------------------------------------------
29
2.4.4
CO
AND
POST-TRANSCRIPTIONAL
REGULATORS
IN
CELL
FATE
DECISIONS
---------------------------------------------------------
32
3
AIMS
OF
THIS
THESIS
----------------------------------------------------------------------------------------------------------
41
4
RESULTS
----------------------------------------------------------------
---------------------------------------------------------
43
4.1
PART
I
------------------------------------------------------------------------------------------------------------------------
43
4.1.1
QKI
IS
CONSERVED
IN
THE
VERTEBRATES
----------------------------------------------------------------------------------------------
43
4.1.2
DEVELOPMENTAL
EXPRESSION
PROFILES
OF
QKI
IN
HUMAN
IN
VITRO
CARDIOMYOCYTE
DIFFERENTIATION
MODEL
-45
4.1.3
SUBCELLULAR
LOCALIZATION
OF
QKI
------------------------------------------------------------------------------------------------------
47
4.1.4
CRISPR/CAS9-MEDIATED
GENERATION
OF
QKI
KNOCKOUT
HESC
LINES
------------------------------------------------------
50
4.1.5
VALIDATION
OF
PLURIPOTENCY
UPON
DEPLETION
OF
QKI
IN
HESCS
-------------------------------------------------------------
52
4.1.6
DECIPHERING
THE
ROLE
OF
QKI
IN
LINEAGE
COMMITMENT
OF
HESCS
--------------------------------------------------------
54
4.1.7
MECHANISTIC
INSIGHTS
INTO
THE
FUNCTION
OF
QKI
DURING
CARDIAC
DIFFERENTIATION
---------------
:
-----------------
66
4.2
PART
II
------------------------------------------------------------------------------------------------
-
-----------------
73
4.2.1
GLOBAL
TRANSCRIPTOMIC
ANALYSIS
DURING
ZEBRAFISH
HEART
REGENERATION
----------------------------------------------
73
4.2.2
IDENTIFICATION
OF
MIRNA-DEPENDENT
GENE
REGULATORY
NETWORK
CONTROLLING
ZEBRAFISH
HEART
REGENERATION
---------------------------------------------------------------------------------------------------------------------------------------------
78
4.2.3
FUNCTIONAL
CHARACTERIZATION
OF
DRE-MIR-429A
DURING
ZEBRAFISH
HEART
REGENERATION
-------------------------
83
5
DISCUSSIONS
-
-
-
-
-
-
-
--
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
88
5.1
PART
I------------------------------------------------------------------------------------------------------------------------88
5.1.1
QKI
IS
AN
EVOLUTIONARIIY
CONSERVED
RBP
ENRICHED
IN
THE
NUCLEUS
DURING
THE
CARDIAC
COMMITMENT
OF
HUMAN
EMBRYONIC
STEM
CELLS
-----------------------------------------------------------------------------------------------------------------
88
5.1.2
QKI
IS
NOT
ESSENTIAL
FOR
THE
INDUCTION
OF
GERM
LAYERS
----------------------------------------------------------------------
89
5.1.3
THE
INTERPLAY
OF
QKI
WITH
WNT
SIGNALING
IS
CRITICAL
FOR
CARDIAC
MESODERM
SPECIFICATION
-------------------
90
5.1.4
QKI
LIKELY
SETS
THE
MRNA
SPLICING
CODE
FOR
CARDIAC
DIFFERENTIATION
AND
CONTRACTILITY
-------------------------91
5.1.5
QKI
DIRECTLY
TARGETS
THE
PRE-MRNA
OF
GENES
INVOLVED
IN
WNT
SIGNALING
AND
CARDIAC
MYOFIBRIL
FORMATION
--------------------------------------------------------------------------------------------------------------------------------------------------
95
5.1.6
CONCLUSION
I
---------------------------------------------------------------------------------------------------------------------------------
97
5.1.7
OUTLOOKS
I
-------------------------------------------------------------------------------------------------------------------------------------98
5.2
PART
II
----------------------------------------------------------------------------------------------------------------------
99
5.2.1
COMBINED
MIRNA AND
MRNA
PROFILING
REVEALS
THAT
HEART
REGENERATION
IS
A
TRANSIENT
CELLULAR
REPROGRAMMING
PROCESS
----------------------------------------------------------------------------------------------------------------------------
99
5.2.2
IDENTIFICATION
AND
RANKING
OF
MIRNA-DEPENDENT
GENE
REGULATORY
NETWORKS
DURING
ZEBRAFISH
HEART
REGENERATION
----------------------------------------------------------------------------------------------------------------------------------
101
5.2.3
THE
DRE-MIR-429A
IS
A
CRUCIAL
REGULATOR
FOR
CM
PROLIFERATION
DURING
ZEBRAFISH
CARDIAC
REGENERATION
--------------------------------------------------------------------------------------------------------------------------------------------
102
5.2.4
CONCLUSION
II
------------------------------------------------------------------------------------------------------------------------------104
5.2.5
OUTLOOKS
II
---------------------------------------------------------------------------------------------------------------------------------
104
6
MATERIAL
AND
METHODS
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
105
6.1
MATERIALS
---------------------------------------------------------------------------------------------------------------
105
6.1.1
CHEMICALS
----------------------------------------------------------------------------------------------------------------------------------
105
6.1.2
ANTIBODIES
.
107
6.1.3
PLASMIDS--------------------------------------------------------------------------------------------------------------------------
*
--------
109
6.1.4
BACTERIAL
STRAINS
---------------------------------------------------------------------------------------------
110
6.1.5
HUMAN
CELL
LINES
.
-
-
-
---------------------
110
6.1.6
MICE
CELL
LINES
----------------------------------------------------------------------------------------------------------------------------110
6.1.7
ZEBRAFISH
LINES
---------------------------------------------------------------------------------------------------------------------------110
6.2
ANIMAL
HUSBANDRY
AND
ANIMAL
EXPERIMENT
----------------------------------------------------------------
111
6.2.1
ZEBRAFISH
HUSBANDRY
------------------------------------------------------------------------------------------------------------------
HL
6.2.2
CARDIAC
INJURY
------------------------------------------------------------
-
--------------
-
------------------------------------
111
6.2.3
INTRA-CARDIAC
INJECTION
OF
MIRNA
INHIBITOR
OR
MO
-----------------------------------------------------------------------
111
6.2.4
ZEBRAFISH
BREEDING
AND
MICROINJECTION
-
-
---------------------------------------------------------------
HL
6.2.5
COLLECTION
OF
THE
HEARTS
POST-INJURY
--------------------------------------------------------------------------------------------
112
6.2.6
PREPARATION
OF
THE
HISTOLOGICAL
SAMPLES
-------------------------------------------------------------------------------------
113
6.3
CELL
CULTURE
-------------------------------------------------------------------------------------------------------------
113
6.3.1
COATING
OF
CELL
CULTURE
DISHES
WITH
MATRIGEL
----------------------------------------------------------------------------------
113
6.3.2
MAINTENANCE
CULTURE
OF
HESCS
-----------------------------------------------------------------------------------------------------
113
6.3.3
FREEZING
OF
HESCS
FOR
LONG-TERM
STORAGE
--------------------------------------------------------------------------------------
114
6.3.4
CARDIOMYOCYTE
DIFFERENTIATION
-----------------------------------------------------------------------------------------------------
114
6.3.5
ADDITIONAL
MESODERM
DIFFERENTIATION
PROTOCOLS
---------------------------------------------------------------------------
115
6.3.6
INDUCTION
OF
ECTODERM
AND
ENDODERM
LINEAGE
DIFFERENTIATION
-----------------------------------------------------
116
6.3.7
GENERATION
OF
CRISPR/CAS9-MEDIATED
KNOCKOUT
OF
GENES
IN
HESCS-----------------------------------------------
116
6.3.8
GENERATION
OF
STABLE
DOXYCYCLINE-INDUCIBLE
QKI
OVEREXPRESSION
LINES
IN
QKI-KO
HESCS
-----------------
117
6.4
MOLECULAR
BIOLOGY
----------------------------------------------------------------------------------
118
6.4.1
BACTERIA
GROWTH
CONDITIONS
---------------------------------------------------------------------------------------------------------
118
6.4.2
TRANSFORMATION
OF
BACTERIA
---------------------------------------------------------------------------------------------------------
118
6.4.3
PLASMID
ISOLATION
-------------------------------------------------------------------------------------------------------------------------
119
6.4.4
PCR
PRIMERS
--------------------------------------------------------------------------------------------------------------------------------
119
6.4.5
POLYMERASE
CHAIN
REACTION
(PCR)
-------------------------------------------------------------------------------------------------
120
6.4.6
COLONY
PCR
---------------------------------------------------------------------------------------------------------------------------------
121
6.4.7
AGAROSE
GEL
ELECTROPHORESIS
--------------------------------------------------------------------------------------------------------
122
6.4.8
GENERATION
OF
CRISPR
PLASMIDS
----------------------------------------------------------------------------------------------------
122
6.4.9
SGRNA
TEMPLATE
ASSEMBLY
AND
PREPARATION
OF
SGRNA
-------------------------------------------------------------------
123
6.4.10
RNA
EXTRACTION
-----------------------------------------------------------------------------------------------------------------------
124
6.4.11
DNASE
TREATMENT
OF
PURIFIED
RNA
--------------------------------------------------------------------------------------------
124
6.4.12
REVERSE
TRANSCRIPTION
--------------------------------------------------------------------------------------------------------------
124
6.4.13
RNA
PRECIPITATION
-------------------------------------------------------------------------------------------------------------------
125
6.4.14
SEMI-QUANTITATIVE
REAL-TIME
PCR
(QPCR)
---------------------------------------------------------------------------------
126
6.4.15
MIRNA
QPCR
--------------------------------------------------------------------------------------------------------------------------
127
6.4.16
ENHANCED
CROSS-LINKING
IMMUNOPRECIPITATION
FOLLOWED
BY SEQUENCING
(ECLIP-SEQ)
-------------------
128
6.5
PROTEIN
BIOCHEMISTRY
----------------------------------------------------------------------------------------------
128
6.5.1
PROTEIN
ISOLATION
-------------------------------------------------------------------------------------------------------------------------
128
6.5.2
SODIUM
DODECYL
SULFATE-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
--------------------------------------
129
6.5.3
WET
BLOTTING
OF
PROTEINS
ON
POLYVINYLIDENE
DIFLUORIDE
(PVDF)
MEMBRANES
-----------------------------------
130
6.5.4
IMMUNOPRECIPITATION
FOR
MASS
SPECTROMETRY
AND
WB
-----------------------------------------------------------------
130
6.5.5
NUCLEAR-CYTOSOLIC
FRACTIONATION
.
131
6.6
HISTOLOGICAL
EXPERIMENT
-------------------------------------------------------------
-
---------------------------
131
6.6.1
SURIS
RED
AND
FAST
GREEN
STAINING
---------------------------
131
6.6.2
IMMUNOCYTOCHEMISTRY
----------------------------------------------------------------------------------------------------------------
132
6.6.3
MITO-TRACKER
STAINING
------------------------------------------------------------------------------------------------------------------
132
6.6.4
ER
TRACKER
STAINING
---------------------------------------------------------------------------------------------------------------------
133
6.7
RTCA
TO
MEASURE
BEATING
ACTIVITY
OF
HESC-CMS
-------------------------------------------------------
133
6.8
BIOINFORMATIC
ANALYSIS
-------------------------------------------------------------------------------------------
134
6.8.1
RNA
SEQUENCING
DATA
ANALYSIS
-------
--------------------------------------------------------------------------------------------
134
6.8.2
PREDICTION
OF
MIRNA
TARGETS
-------------------------------------------------------------------------------------------------------
134
6.8.3
IDENTIFICATION
OF
MIRNA-DEPENDENT
NETWORKS
REGULATING
HEART
REGENERATION
--------------------------------
134
6.8.4
PROTEOMIC
DATA
ANALYSIS
-------------------------------------------------------------------------------------------------------------
134
6.8.5
ECLIP
ANALYSIS
-----------------------------------------------------------------------------------------------------------------------------
134
6.9
DATA
ANALYSIS
AND
PLOTTING
-------------------------------------------------------------------------------------135
6.9.1
STATISTICAL
ANALYSIS
----------------------------------------------------------------------------------------------------------------------
135
6.9.2
CORRELATION
ANALYSIS
--------------------------------------------------------------------------------------------------------------------
135
6.9.3
PRINCIPAL
COMPONENT
ANALYSIS
(PCA)
--------------------------------------------------------------------------------------------
135
6.9.4
HEATMAPS
--------------------------------------------------------------------------------------------------------------------------------
~
135
6.9.5
CIRCLE
PLOTS
----------------------------------------------------------------------------------------------------------------------------------
135
6.9.6
GO
ENRICHMENT
ANALYSIS
-------------------------------------------------------------------------------------------------------------
135
7
REFERENCES
------------------------------------------------
136
8
ACKNOWLEDGEMENTS
-
172
9
ERKLARUNG
ZUR
DISSERTATION
-------------------------------------------------------------------------------------------
174 |
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| author | Yao, Wenjie |
| author_GND | (DE-588)1217441034 |
| author_facet | Yao, Wenjie |
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| ctrlnum | (OCoLC)1249677836 (DE-599)DNB1222596768 |
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| discipline_str_mv | Medizin |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV047833612 |
| illustrated | Illustrated |
| index_date | 2024-07-03T19:09:42Z |
| indexdate | 2024-07-10T09:22:36Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-033216832 |
| oclc_num | 1249677836 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | 174, 1-2 Seiten Illustrationen, Diagramme 21 cm |
| publishDate | 2020 |
| publishDateSearch | 2020 |
| publishDateSort | 2020 |
| record_format | marc |
| spelling | Yao, Wenjie Verfasser (DE-588)1217441034 aut RNA regulons during heart development and regeneration Wenjie Yao Köln [2020] 174, 1-2 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2020 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1222596768/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033216832&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p aepkn 0,95837 20201211 DE-101 https://d-nb.info/provenance/plan#aepkn 2\p dnb 20211104 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Yao, Wenjie RNA regulons during heart development and regeneration |
| subject_GND | (DE-588)4113937-9 |
| title | RNA regulons during heart development and regeneration |
| title_auth | RNA regulons during heart development and regeneration |
| title_exact_search | RNA regulons during heart development and regeneration |
| title_exact_search_txtP | RNA regulons during heart development and regeneration |
| title_full | RNA regulons during heart development and regeneration Wenjie Yao |
| title_fullStr | RNA regulons during heart development and regeneration Wenjie Yao |
| title_full_unstemmed | RNA regulons during heart development and regeneration Wenjie Yao |
| title_short | RNA regulons during heart development and regeneration |
| title_sort | rna regulons during heart development and regeneration |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1222596768/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033216832&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT yaowenjie rnaregulonsduringheartdevelopmentandregeneration |
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