°C Degree celsius
µ Micro
µg Microgram
a.u. Arbitrary units
aa Amino acids
AFP Alpha-1-fetoprotein
ALB Albumin
APD Action potential duration
Approx. Approximately
APS Ammonium persulfate
ATP Adenosine triphosphate
ASE Allele-specific expression
BafA1 Bafilomycin A1
bp Base pair
BSA Bovine serum albumin
Ca2+ Calcium
CaM Calmodulin
CamKII Ca2+/Calmodulin-dependent protein kinase II cAMP Cyclic adenosine monophosphate
Cas9 CRISPR-associated protein 9
CASQ2 Calsequestrin 2
cDNA Complementary DNA
CM Cardiomyocyte
c-MYC V-myc myelocytomatosis avian viral oncogene homolog
CO2 Carbon dioxide
CPVT Catecholaminergic polymorphic ventricular tachycardia CRISPR Clustered regularly interspaced short palindromic repeats
cTNT Cardiac troponin T
Cx43 Connexin 43
DAD Delayed afterdepolarization
DAPI 4′, 6-Diamidino-2-phenylindole dihydrochloride ddH2O Double distilled water
DMEM Dulbecco's modified eagle medium
126
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic Acid
DPBS Dulbecco's phosphate buffered saline
DSB Double strand break
DTT Dithiothreitol
E. coli Escherichia coli
EAD Early afterdepolarization
ECL Enhanced chemiluminescence
EDTA Ethylene diamine tetra acetic acid ESCs Embryonic stem cells
F Fluorescence
FACS Fluorescence-activated cell sorting
FBS Fetal bovine serum
FDHM Full duration at half maximum
Fig. Figure
FITC Fluorescein isothiocyanate
FL Feeder layer
for Forward
FOXD3 Forkhead box D3
FSC Forward scatter
FWHM Full width at half maximum
g Gram
GAPDH Glyceraldehyde 3-phosphate dehydrogenase GDF3 Growth differentiation factor 3
gDNA Genomic DNA
h Hours
hbFGF Fibroblast growth factor basic
HDR Homology directed repair
HEK293 Human embryonic kidney 293
HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid hERG Human ether-à-go-go-related gene
hESC Human embryonic stem cell
hiPSC Human induced pluripotent stem cell
HR Homologous recombination
HRP Horseradish peroxidase
Hz Hertz
IP3R Inositol 1,4,5-trisphosphate receptors
127
IRX4 Iroquois homeobox 4
Iso Isoprenaline
KCl Potassium chloride
kDa Kilodalton
KIR2.1 Inward rectifier potassium channel 2 KLF4 Krüppel-like factor 4
l Liter
mA Milliampere
MEA Multi-electrode array
MEFs Mouse embryonic fibroblasts
min Minutes
ml Milliliter
MLC2A Myosin light chain 2a MLC2V Myosin light chain 2 v MOI Multiplicity of infection
ms Milliseconds
MSC Mesenchymal stem cell
MTG Monothioglycerol
n Nano
Na2HPO4 Disodium hydrogen phosphate
NaOH Sodium hydroxide
Nav1.5 Cardiac voltage-gated Na+ channel
NCX Sodium-calcium exchanger
NEAA Non-essential amino acids
OCT4 Octamer binding transcription factor 4 OCT4 Octamer-binding transcription factor 4 PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
PDE4D3 Phosphodiesterase 4D3
PE Phycoerythrin
pF Picofarad
PFA Paraformaldehyde
pH Negative logarithmic value of the H+ concentration
PLB Phospholamban
PP1 Protein phosphatase 1
PP2A Protein phosphatase 2a
PTC Premature termination codon
128
rev Reverse
rpm Revolutions per minute
RPMI Roswell Park Memorial Institute
RT Room temperature
RYR Ryanodine receptor
SDS Sodium sodecyl sulfate
sec Seconds
SEM Standard error
Ser Serine
SERCA Sacro/ endoplasmic reticulum Ca2+-ATPase
SeV Sendai virus
SMA Smooth muscle actin
SOX2 Sex determining region Y box 2 SOX2 Sex Determining Region Y-Box 2
SR Sacroplasmic reticulum
SSEA4 Stage-specific embryonic antigen-4 ssODNs Single-stranded oligodeoxynucleotides STEMCCA Stem cell cassette
SYP Synaptophysin
Tab. Table
TALEN Transcription activator-like nuclease TBE buffer Tris-borate EDTA buffer
TBS Tris buffered saline
TBS-T Tris buffered saline with Tween-20
TC Tissue culture
TEMED Tetramethylenediamine
TH Tyrosine hydroxylase
Thr Threonine
TRA-1-60 Tumor rejection antigen 1-60
Tris Tris-(hydroxymethyl)-aminomethane
TZV Thiazovivin
U Unit
V Voltage
v Volume
VCAM-1 Vascular cell adhesion molecule 1
VT Ventricular tachycardia
ZFN Zinc finger nuclease
129
α Alpha
α-ACT α-Actinin
α-MHC Myosin heavy chain α isoform
β Beta
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7.2 List of Figures
Figure 1. Ca2+-induced Ca2+ release and triggered arrhythmias... 5
Figure 2. Structure of RYR2 including hot-spot regions of mutations. ... 6
Figure 3. Generation of patient-specific hiPSCs for disease modeling and drug screening. 9 Figure 4. Key steps sufficient for cardiac differentiation of hiPSCs. ... 16
Figure 5. Genome editing using CRISPR/Cas9. ... 18
Figure 6. CRISPR/Cas9 plasmid containing the sequence of gRNA, Cas9, GFP and kanamycin. ... 23
Figure 7. RYR2 protein and mutations studied in the present study. ... 54
Figure 8. Timeline for the generation of CPVT-hiPSCs. ... 55
Figure 9. Morphology and alkaline phosphatase activity of CPVT-hiPSCs. ... 55
Figure 10. Gene expression analysis of generated CPVT-hiPSCs and their parental fibroblasts. ... 56
Figure 11. Immunostaining detects pluripotency-related proteins of generated CPVT-hiPSCs... 57
Figure 12. Gene expression of differentiated CPVT-hiPSCs. ... 59
Figure 13. Immunostaining of differentiated hiPSCs detects germ layer-specific proteins. ... 60
Figure 14. Teratoma formation of CPVT-hiPSCs. ... 61
Figure 15. Verification of the RYR2 point mutation in the generated CPVT-hiPSCs. ... 62
Figure 16. Schematic illustration of the directed cardiac differentiation using hiPSCs. ... 63
Figure 17. Flow cytometric analysis of 3-month-old CMs. ... 63
Figure 18. Expression of cardiac-specific marker genes. ... 64
Figure 19. Cardiac differentiation of CPVT-hiPSCs. ... 65
Figure 20. Expression of CX43 in CPVT-CMs. ... 66
Figure 21. Patch clamp analysis of CPVT- and Ctrl-CMs. ... 67
Figure 22. Basal and isoprenaline-induced Ca2+ sparks in CPVT- and Ctrl-CMs. ... 68
Figure 23. Allele-specific RYR2 expression of CPVT-CMs. ... 69
Figure 24. RYR2 expression in CPVT- and Ctrl-CMs. ... 70
Figure 25. Phospho-RYR2 expression in CPVT- and Ctrl-CMs. ... 71
Figure 26. Cleavage assay of CRISPR/Cas9-transfected cells. ... 72
Figure 27. Strategy for the generation of CRISPR/Cas9-targeted hiPSCs. ... 73
131
Figure 28. Screening of CRISPR/Cas9-transfected cell clones. ... 74
Figure 29. Sequencing of CRISPR/Cas9-targeted clones. ... 75
Figure 30. Gene expression of Ca2+ regulating genes in CRISPR/Cas9-edited CMs. ... 76
Figure 31. Allele-specific RYR2 expression in RYR2+/Ø-T42-CMs. ... 77
Figure 32. RYR2 expression in CRISPR/Cas9-edited CMs. ... 78
Figure 33. Immunostaining of RYR2Ø/Ø-A3-CMs in comparison to CPVTc2.1-CMs. ... 79
Figure 34. Analysis of RYR2 protein degradation in CRISPR/Cas9-edited CMs. ... 80
Figure 35. Expression of Ca2+ regulatory proteins in RYR2Ø/Ø-A3-CMs. ... 80
Figure 36. Isoprenaline-induced Ca2+ sparks in CRISPR/Cas9-engineered CMs. ... 81
Figure 37. Ca2+ transients from CRISPR/Cas9-engineered CMs. ... 82
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7.3 List of Tables
Table 1: List of laboratory equipment ... 21
Table 2: List of disposable items... 22
Table 3: List of CRISPR/Cas9-plasmids. ... 24
Table 4: List of oligonucleotides for reverse transcription-PCR analysis ... 24
Table 5: Single-stranded oligodesoxynucleotides (ssODNs) used for CRISPR/Cas9 technology. ... 25
Table 6: List of oligonucleotides used for screening of CRISPR/Cas9-edited clones ... 26
Table 7: List of oligonucleotides for allele-specific sequencing ... 26
Table 8: List of primary antibodies ... 27
Table 9: List of secondary antibodies ... 28
Table 10: List of molecular biological reagents ... 29
Table 11: List of components for molecular biological methods, and protein analyses .... 31
Table 12: List of competent E. coli cells ... 32
Table 13: List of components for cell culture ... 33
Table 14: List of buffers and solutions for cell culture ... 34
Table 15: List of media for human cell lines ... 35
Table 16: hiPSC lines used in this work and their specifications ... 36
Table 17: List of software used for analysis ... 37
Table 18: List of components for reverse transcription reaction ... 43
Table 19: List of components for PCR ... 43
Table 20: PCR for the amplification of cDNA used for allele-specific expression analysis . 46 Table 21: Components for 12 ml separation gel and 7.5 ml stacking gel ... 47
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7.4 Curriculum Vitae
Personal details
Name Sarah Henze
Date of birth 06/11/1988
Place of birth Göttingen, Germany
Nationality German
Address Falkenhorst 12, 34346 Hann. Münden Education
Since Mai 2013 PhD study of Molecular Medicine at the Department of Cardiology and Pneumology, Georg-August University Göttingen
PhD Thesis: Induced pluripotent stem cell-derived cardiomyocytes as model for studying CPVT caused by mutations in RYR2
10/2010 - 03/2013 Master of Science study of “Developmental, Neural and Behavioral Biology” at the Georg-August University Göttingen
Master thesis: Chemical-versus Ligand-induced Heterodimerization of β-Arrestin and Chemokine Receptors CXCR4/CCR5
10/2007 -10/2010 Bachelor of Science, Molecular Biosciences, at the Georg-August-University Göttingen
Bachelor thesis: “Identifizierung der Interaktionspartner der S-Adenosylmethioninsynthetase unter der Verwendung der Tandem-Affinitäts-Aufreinigung in Aspergillus nidulans”
2004-2007 “Berufliches Gymnasium”, Specialization: Biotechnology, Witzenhausen
2000-2004 “Werra-Realschule”, Hann. Münden
1994-2000 Primary School “Königshof” and “Orientierungsstufe I”, Hann. Münden