| Abstract | 第1-8页 |
| 中文摘要 | 第8-10页 |
| Abbreviations | 第10-12页 |
| Chapter 1 Background of KCTD10 gene | 第12-22页 |
| ·Characterization of KCTD10 and family members | 第12-14页 |
| ·Structure analysis of KCTD10 | 第14-15页 |
| ·KCTD10 expression in adult tissues | 第15-16页 |
| ·The regulation of KCTD10 | 第16-17页 |
| ·The functions of KCTD10 | 第17-18页 |
| Reference | 第18-22页 |
| Chapter 2 Mouse KCTD10 cloning and expression pattern during the early embryogenesis | 第22-28页 |
| ·Introduction | 第22页 |
| ·Materials and methods | 第22-25页 |
| ·RNA preparation and reverse transcription | 第22页 |
| ·Plasmids construction | 第22-23页 |
| ·Embryo preparation for whole mount in situ hybridization | 第23页 |
| ·Digoxigenin-UTP-labeled RNA probes preparation | 第23-24页 |
| ·Whole mount in situ hybridization | 第24-25页 |
| ·Result and discussion | 第25-27页 |
| ·Sequence analysis of mouse KCTD10 | 第25页 |
| ·KCTD10 expression in the early stages of development | 第25-27页 |
| Reference | 第27-28页 |
| Chapter 3 Generation of KCTD10 conventional knockout mice | 第28-46页 |
| ·Introduction | 第28页 |
| ·Materials and methods | 第28-38页 |
| ·Targeting vector design and construction | 第28-29页 |
| ·Preparation of feeder cell layers from MEF cells | 第29页 |
| ·Embryonic stem (ES) cell culture | 第29-30页 |
| ·Electroporation of targeting vector DNA into ES cell | 第30-31页 |
| ·Isolation of ES cell colonies | 第31页 |
| ·Freezing ES cells in 96-well plates | 第31-32页 |
| ·Screening positive ES colonies | 第32-34页 |
| ·Thawing positive ES cells from a 96-well plate | 第34-35页 |
| ·Production of chimeras | 第35页 |
| ·Generation of KCTD10 conventional knockout mice | 第35页 |
| ·Genotyping | 第35-36页 |
| ·RNA extraction | 第36-37页 |
| ·Reverse transcription and real-time PCR check the mRNA levels | 第37页 |
| ·Protein extraction | 第37页 |
| ·Antibody against KCTD10 preparation | 第37页 |
| ·Western blot | 第37-38页 |
| ·Results | 第38-44页 |
| ·Targeting vector design and construction | 第38-40页 |
| ·Screen of homologous recombined ES cell colonies by PCR | 第40页 |
| ·Identification of homologous recombined ES cell clones by southern blot | 第40-41页 |
| ·Generation of KCTD10 mutant mice | 第41-44页 |
| ·Discussion | 第44-45页 |
| Reference | 第45-46页 |
| Chapter 4 Phenotypes of KCTD10-deficient mouse in cardiovascular system | 第46-56页 |
| ·Introduction | 第46-47页 |
| ·Materials and methods | 第47-48页 |
| ·Mice breeding and Embryos preparation | 第47页 |
| ·Histological analysis | 第47-48页 |
| ·Whole mount immunostaining | 第48页 |
| ·Immunofluorescence | 第48页 |
| ·Results | 第48-53页 |
| ·KCTD10 disruption in mice causes embryonic death | 第48-49页 |
| ·KCTD10 deficient embryos showed angiogenesis defects | 第49-53页 |
| ·KCTD10 deficient embryos showed heart defects | 第53页 |
| ·Discussion | 第53-54页 |
| Reference | 第54-56页 |
| Chapter 5 Notch signaling was affected by the KCTD10 disruption | 第56-70页 |
| ·Introduction | 第56-57页 |
| ·Materials and methods | 第57-60页 |
| ·Cell culture and siRNA transfection | 第57-58页 |
| ·Western blot | 第58页 |
| ·Endogenous immunoprecipitation (IP) assay | 第58页 |
| ·Immunofluorescent staining | 第58-59页 |
| ·RNA preparation, semi quantitative real-time PCR and quantitative real-time PCR | 第59-60页 |
| ·Results | 第60-64页 |
| ·KCTD10 is induced by VEGF in a dose-and time-dependent manner | 第60-61页 |
| ·Some key members in Notch signaling were upregulated in KCTD10-/ homozygous embryos | 第61-63页 |
| ·KCTD10 interacts with Notch1 and Cul3 simultaneously | 第63页 |
| ·KCTD10 decreases the protein stability of Notch1 | 第63-64页 |
| ·Discussion | 第64-66页 |
| Reference | 第66-70页 |
| Chapter 6 The functions of Notch signaling in the cardiovascular system | 第70-90页 |
| ·The core Notch pathway | 第70-72页 |
| ·The functions of Notch signaling in the vascular development | 第72-73页 |
| ·The functions of Notch signaling in the heart development | 第73-76页 |
| ·Cardiomyocyte differentiation | 第74-75页 |
| ·Atrioventricular canal development | 第75页 |
| ·Valve development | 第75-76页 |
| ·Ventricular trabeculation | 第76页 |
| ·Outflow tract remodeling | 第76页 |
| ·Conclusion | 第76-77页 |
| Reference | 第77-90页 |
| Acknowledgments | 第90-92页 |
| Publication | 第92-95页 |
