臺灣博碩士論文加值系統

 MicroRNA (miRNA) regulates gene expression by silencing target mRNAs, and is critical for various developmental processes including organogenesis. However, the regulatory networks of miRNAs in the development of different organs remain largely elusive, partly due to the technical difficulties in identifying organ-specific miRNAs. In particularly, the isolation of tissue-specific cells and associated miRNA is challenging due to the small size and tissue complexity in the developing organs like heart. Conventionally, fluorescence-activated cell sorting (FACS) is used to separate fluorescent cardiomyocytes (CM) from transgenic fish with a fluorescent heart. However, the FACS requires large number of embryos and tedious sorting procedures that often leads to cell damage and more critically results in the changes in miRNA/mRNA landscape. To address this question, I have established a novel method to isolate heart-specific miRNAs by Tandem Affinity Purification (TAP) of Argonaute 2 in zebrafish embryos. Using a binary Gal4-UAS system, our laboratory had previously generated a transgenic fish expressing GLUE-tagged Argonaute 2 (Ago2) driven by a UAS promoter, named Tg (UAS:H2A-mCherry-p2A-GLUE-ago2), and then it was crossed to a cardiac-specific Gal4 line, Tg (myl7:gal4) to become a double-transgenic fish expressing GLUE-Ago2 specifically in CMs. The Ago2 is a key component of the RNA-induced silencing complex which binds to mature miRNAs, while the GLUE contains affinity domains for TAP.
In this thesis, I optimized the purification procedures of TAP and successfully purified the GLUE-Ago2 protein with the associated miRNAs from zebrafish CMs at 24 h and 48 post fertilization (hpf), which are critical stages for zebrafish cardiac development. The Ago2-associated miRNAs were profiled by next generation sequencing (NGS). For technical comparison, I also collected CMs from the same transgenic line by FACS for miRNA profiling. NGS data revealed that currently known CM-specific miRNAs were identified by both methods, but the TAP appears to be superior because: (1) It requires only a quarter of the sample input compared to the FACS; (2) It has higher reads in low abundant miRNAs; (3) It only isolates mature miRNAs whereas the FACS obtains all small RNAs. Target gene prediction, gene ontology, and pathway analysis further revealed both known and potentially important regulatory networks in CM differentiation and maturation. Using quantitative real-time PCR, I found the expression of selected miRNAs with higher fold changes between 24 and 48 hpf was similar to their respective expression in FACS but much lower in TAP NGS data. The discrepancy will be discussed. The potential roles of candidate miRNAs and target genes in cardiogenesis will be evaluated in the future. Taken together, I demonstrate that the TAP is a valuable tool to identify those differentially expressed cardiac-specific miRNAs during development. This method can thus be potentially extended to study other tissues or organs with appropriate GAL4 driver available.

中文摘要 1
Abstract 2
Introduction 5
Biogenesis of microRNA 5
Roles of miRNAs in Cardiac Development and Function 6
Tandem affinity purification methods and application 9
Zebrafish as a model for cardiovascular research 11
Objectives of this study 12
Materials and Methods 13
Zebrafish maintenance and embryos collection 13
Generation of transgenic zebrafish lines 13
Plasmids constructions 14
Cell culture and transfection 14
Tandem-affinity purification of the Glue-Ago2 complex 15
Preparation of Glue-Ago2 protein 17
Western blot 17
Fluorescence-activated cell sorting (FACS) 18
Construction of cDNA libraries, sequencing, and analysis (NGS) 19
Statistical analysis 20
Results 21
Expression of the pGlue vector containing Ago2 and Tandem-Affinity Purification (TAP) of Ago2 in HEK293T cells 21
Generation, screening and validation of Tg (myl7: Gal4; UAS:H2A mCherry-GSG2A-Glue-Ago2) fish 22
Purification of Ago2 from Tg(GlueAgo2) embryos by Tandem-Affinity Purification 24
Sorting of mCherry-expressing heart cells by flow cytometry 27
Presence of cardiac-enriched miRNAs in samples collected by FACS and TAP 28
MicroRNAs profiling in TAP and FACS 29
Low sample variations between duplicates 30
MicroRNAs expression profile 31
Target gene prediction and functional annotation 32
Enrichment of mature functional miRNAs by TAP 34
Validation of candidate cardiac miRNAs expression 35
Discussion 36
References 42
Tables 48
Figures 63

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