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CRISPR/CAS system을 이용한 돼지 단위발생 배아에서 Oct-4 유전자의 삭제와 삽입 / 권정우
15 유전학 조회 수 142 추천 수 0 2017.02.03 14:42:36| 발행년 : | 2015 |
|---|---|
| 구분 : | 학위논문 |
| 학술지명 : | |
| 관련링크 : | http://www.riss.kr/link?id=T13697155 |
- 기타서명
Oct-4 genes knock-out and knock-in porcine parthenogenetic embryos using CRISPR/Cas9 system
- 저자
권정우
- 형태사항
vi, 46 p. : 삽화 ; 26 cm.
- 일반주기
충북대학교 논문은 저작권에 의해 보호됩니다
指導敎授: 金南衡
참고문헌: p.34-44 - 학위논문사항
학위논문(석사)-- 忠北大學校 一般大學院 : 畜産·園藝·食品工學科 畜産學專攻 2015. 2
- KDC
527.346 5
- 발행국
충청북도
- 언어
영어
- 출판년
2015
초록 (Abstract)
- The CRISPR/Cas9 system has proven to be an efficient gene-editing tool for genome modification of cells and organisms. However, the applicability and efficiency of this system in pig embryos have not been studied in depth. Here, we aimed to remove porcine OCT4 function as a model case using the CRISPR/Cas9 system. Injection of Cas9 and single-guide RNA (sgRNA) against OCT4 decreased the percentages of OCT4-positive embryos to 37–50% of total embryos, while ~100% of control embryos exhibited clear OCT4 immunostaining. We assessed the mutation status near the guide sequence using polymerase chain reaction (PCR) and DNA sequencing, and a portion of blastocysts (20% in exon 2 and 50% in exon 5) had insertions/deletions near protospacer-adjacent motifs (PAMs). Different target sites had frequent deletions, but different concentrations of sgRNA made no impact. OCT4 mRNA levels dramatically decreased at the 8-cell stage, and they were barely detectable in blastocysts, while mRNA levels of other genes, including SOX2, NANOG, and CDX2 were not affected. In addition, the combination of two sgRNAs led to large-scale deletion (about 1.8 kb) in the same chromosome. Next, we injected an enhanced green fluorescent protein (eGFP) vector targeting the OCT4 exon with Cas9 and sgRNA to create a knock-in. We confirmed eGFP fluorescence in blastocysts in the inner cell mass, and also checked the mutation status using PCR and DNA sequencing. A significant portion of blastocysts had eGFP sequence insertions near PAM sites. The CRISPR/CAS9 system provides a good tool for gene functional studies by deleting target genes in the pig.
- Ⅰ. Introduction 1
- Ⅱ. Literature Review 4
- 1. Genome editing 4
- 2. CRISPR/Cas9 system 5
- 3. OCT-4 genes(Octamer-binding transcription factor 4) 6
- 4. Pre-implantation embryo development and maternal to zygotic transition 7
- Ⅲ. Materials and Methods 9
- 1. Chemicals 9
- 2. Generation of Cas9 mRNA and sgRNAs 9
- 3. Generation of OCT4-green fluorescent protein (GFP) knock-in constructs 10
- 4. In vitro porcine oocyte maturation 11
- 5. Parthenogenetic activation and in vitro culture (IVC) 11
- 6. Cas9/sgRNA injections in porcine parthenogenetic zygotes 12
- 7. Preparation of genomic DNA from blastocysts and PCR amplification 13
- 8. Real-time reverse transcription-PCR 13
- 9. Immunofluorescence staining and confocal microscopy 14
- Ⅳ. Results 15
- 1. CRISPR/Cas9-mediated targeting of the OCT4 gene in porcine zygotes 15
- 2. Large-scale genomic region deletion in the OCT4 gene using two sgRNA pairs 16
- 3. Generation of OCT4-GFP knockins using homology-dependent repair 17
- Ⅴ. Discussion 30
- Ⅵ. 요약 33
- Ⅶ. Reference 34
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