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CRISPR-Cas9을 이용한 살아있는 세포에서의 mRNA와 단백질의 동시 시각화 기법

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Abstract
mRNA and proteins play critical roles as spatiotemporal steps maintaining cell homeostasis and regulating biosynthesis. Proteins are important as catalytic materials for various chemical reactions in cells. Thus, detecting mRNA and protein is necessary to understand their regulation. However, there is currently no technology to simultaneously image mRNA and protein in living cells. In this study, a heat shock transcription factor (HSF) 1 donor DNA knock-in cell line was developed using CRISPR-Cas9 to image HSF1 mRNA and HSF1 protein at the same time. A HSF1 knock-in cell line was prepared by inserting donor DNA by homologous recombination around the HSF1 stop codon. Specifically, HSF1 donor DNA contained homologous arms of appropriate length on both sides. EGFP was used for protein tagging, and a puromycin resistance cassette was the selection marker. I also applied the MS2 RNA-labeling system in donor DNA. Live imaging of mRNA dynamics at a single RNA molecule resolution was achieved in live cells using this binary system to express mRNA of the target tagged with 12 copies of RNA hairpin MS2-binding site and fluorescent MS2 bacteriophage coated protein (MCP). In addition, I designed single-guide RNA with low off-target and high cleavage efficiency. A stable HSF1 cell line was constructed using CRISPR-Cas9, and the HSF1 protein was visualized through an enhanced green fluorescent protein signal. Furthermore HSF1 mRNA was visualized in the cytoplasm by exogenously expressing fluorescent MCPs. This system provides a new technique for visualizing specific mRNA and protein to study dynamic changes in endogenous mRNAs and proteins in living cells.
Author(s)
양진욱
Issued Date
2018
Awarded Date
2019-02
Type
Dissertation
Keyword
CRISPR-Cas9
URI
https://oak.ulsan.ac.kr/handle/2021.oak/6161
http://ulsan.dcollection.net/common/orgView/200000172258
Alternative Author(s)
Jinwook Yang
Affiliation
울산대학교
Department
일반대학원 의학과의과학전공
Advisor
김용섭
Degree
Master
Publisher
울산대학교 일반대학원 의학과의과학전공
Language
eng
Rights
울산대학교 논문은 저작권에 의해 보호받습니다.
Appears in Collections:
Medical Science > 1. Theses (Master)
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