Development of fish specific short interfering RNA expression vector and application for RNA interference-mediated suppression of virus replication
- Alternative Title
- 어류 특이적 siRNA 발현 벡터 개발 및 ＲＮＡ 간섭을 통한 바이러스 증식 억제에의 적용
- Abstract
- 이중가닥 RNA (dsRNA)는 특이적으로 mRNA를 분해시키는 세포의 메커니즘인 RNA interference (RNAi) 의 유도 물질로서, 진핵생물의 세포내에서 dsRNA는 dicer라고 하는 효소에 의해 절단 되어 siRNA라고 하는 약 21bp의 작은 RNA가 된다. siRNA는 여러 종류의 단백질과 RNA induced silencing complex (RISC)라는 복합체를 형성해 상보적인 염기서열을 가진 ｍRNA를 특이적으로 분해한다. RNAi를 인위적으로 유도하는 방법으로는 화학적으로 합성된 siRNA를 이용하는 방법과 siRNA를 발현시키는 plasmid 및 virus 벡터를 이용하는 방법의 2가지가 있다. 그러나, 포유류에서 RNAi가 유전자 분석 및 질병의 치료에 널리 응용되고 있는 것에 비해 어류에서는 현재까지 그 보고가 적다. 따라서 본 연구는 어류에서 RNAi를 효과적으로 유도하기 위해 어류의 promoter를 이용한 RNAi 유도 벡터를 구축하였다.
1. 돌돔의 Mx 및 PKR 유전자의 클로닝
dsRNA는 type I interferon (IFN) 반응을 유도하기도 하므로, RNAi의 정확한 분석을 위해서는, type I IFN에 의해 유도되는 여러가지 유전자의 발현 또한 분석하여 결과를 정확하게 판단하는 것이 필요하다. 따라서 본 연구에서는 먼저 type I IFN으로 유도되는 대표적인 유전자인 Mx 및 dsRNA inducible eukaryotic initiation factor 2 kinase (PKR) 유전자를 돌돔으로부터 클로닝하고, dsRNA에 의해 발현이 증가하는 것을 확인하였다. 또한 EGFP를 발현하는 벡터와 dsRNA를 동시에 돌돔에게 주사하였을 때 EGFP의 발현이 비특이적으로 억제되었는데 이는 dsRNA 에 의해 증가된 type I IFN 반응 때문인 것으로 여겨졌다.
2. 어류의 promoter를 이용한 siRNA 발현 벡터의 제작
자주복의 U6 promoter 및돌돔의 β-actin promoter 를 이용하여 siRNA를 short-hairpin RNA (shRNA) 형태로 발현시킬 수 있는 벡터를 제작하여, 어류 주화 세포에서 발현시킨 결과 두 벡터 모두 표적 유전자의 발현을 특이적으로 억제하였다. 또한 두가지 promoter를 비교한 결과β-actin promoter가 보다 더 효과적으로 shRNA를 발현시켜 RNAi를 유도하였다.
3. siRNA 발현 벡터를 이용한 virus 감염의 저해
위 벡터 시스템을 이용하여, 돌돔의 iridovirus증의 원인 바이러스인 rock bream iridovirus (RBIV)의 major capsid protein (MCP) gene을 표적으로 한 siRNA 발현 벡터를 제작하였다. 이를 grunt fin (GF) 세포에 transfection 한 후 RBIV를 접종 하였을 때 대조구에 비해 세포의 생존률이 증가하였으며, MCP 유전자의 발현이 감소한 것을 semi-quantitative RT-PCR을 이용하여 분석하였다. 또한 RBIV의 항 RNAi protein이라 여겨지는 RNase III 유전자의 특성을 조사하였다.
Double-stranded RNA (dsRNA) derived from a replication intermediate of many viruses is one of the major pathogen-associated molecular patterns (PAMP) which triggers innate immune response in vertebrate cells. Furthermore, dsRNA is now also has been paid much attention as a triggering molecule of RNA interference (RNAi), a cellular mechanism which can specifically degrade mRNA. Despite of the robust use of RNAi technique for gene analysis and therapeutics in mammalians, relatively a few studies have reported the use of RNAi in fish species. Thus, in the present study, we aimed to understand the role of dsRNA in the context of exogenous gene expression and to establish effective methods for induction of specific RNAi in fish species.
We first cloned and characterized Mx gene and dsRNA inducible eukaryotic initiation factor 2 kinase (PKR) gene of rock bream (Oplegnathus fasciatus) which are typical type I interferon inducible genes in mammalians and fish. Rock bream Mx gene has been proven to consist of three isoforms, and they are differently up-regulated upon synthetic dsRNA poly I:C stimulation. Rock bream PKR has also been shown to be up-regulated upon dsRNA stimulation. Using rock bream Mx and PKR genes as an indicator of type I interferon response, effect of dsRNA injection on expression of exogenous gene was examined using EGFP reporter gene. As a result, EGFP expression was non-specifically suppressed by strong type I IFN response and specific RNAi effect was also observed but much weaker than non-specific of type I IFN response.
Next, siRNA expression vectors utilizing U6 promoter of fugu (Takifugu rubripes) and β-actin promoter of rock bream modified for effective expression of siRNA have been developed. As a result, both fugu U6 promoter and modified rock bream β-actin promoter could express siRNA as a form of short-hairpin RNA and specifically inhibit EGFP reporter gene expression in fish cell lines. Furthermore, comparison of effectiveness of both promoters has revealed that β-actin promoter could express more efficiently siRNA than fugu U6 promoter.
Finally, we tested possible use of modified rock bream β-actin promoter driven siRNA expression vector for use in inhibition of rock bream iridovirus (RBIV) infection in cell culture system. Grunt fin (GF) cells transfected with β-actin promoter driven siRNA expression vector targeting major capsid protein (MCP) gene of virus exhibited better cell survival upon RBIV infection when compared with control groups. Furthermore, RT-PCR analysis revealed that expression of MCP gene was specifically suppressed in transfected cells. In addition to inhibition experiment of RBIV infection via siRNA expression vector, we have characterized RNase III gene of RBIV which is supposed to be play a role in virus defense mechanism against RNAi.
- Issued Date
- 2009
- Awarded Date
- 2009. 2
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 수산생명의학과
- Advisor
- 김기홍
- Table Of Contents
- General introduction = 1
Chapter Ⅰ. Molecular cloning and characterization of type I interferon inducible Mx genes of rock bream (Oplegnathus fasciatus) = 7
1. Introduction = 8
2. Materials and methods = 10
2.1. Fish = 10
2.2. Initial isolation of partial cDNA sequences of rock bream Mx = 10
2.3. Cloning of full length cDNA sequence and genomic DNA sequence of rock bream Mx = 11
2.4. Analysis of sequence = 12
2.5. Southern blot analysis = 12
2.6. Expression analysis of rock bream Mx isoforms = 13
3. Results = 15
3.1. Cloning and characterization of rock bream Mx isoforms = 15
3.2. Comparison of rock bream Mx isoforms with other fish Mx = 19
3.3. Southern blot analysis of rock bream Mx = 20
3.4. Alternative splicing variants of rock bream Mx isoforms = 21
3.5. Expression analysis of rock bream Mx isoforms = 23
4. Discussion = 27
Chapter Ⅱ. Molecular cloning of rock bream PKR and the effect of long dsRNA on exogenous gene expression in rock bream = 31
1. Introduction = 32
2. Materials and methods = 34
2.1. Fish = 34
2.2. Complementary DNA cloning of rock bream PKR = 34
2.3. Sequence analysis = 35
2.4. Expression analysis of rock bream PKR after poly I:C injection = 36
2.5. dsRNA preparation = 36
2.6. Co-injection of dsRNA and EGFP expression plasmid = 37
2.7. Semi-quantitative RT-PCR analysis = 38
2.8. Statistical analyses = 39
3. Result = 40
3.1. Identification of rock bream PKR = 40
3.2. Comparison of rock bream PKR with other PKRs = 43
3.3. Up-regulation of rock bream PKR after poly I:C injection = 45
3.4. Effect of dsRNA injection on EGFP reporter gene expression = 47
4. Discussion = 50
Chapter Ⅲ. Construction of short interfering RNA (siRNA) expression vector driven by fugu U6 promoter = 55
1. Introduction = 56
2. Materials and methods = 58
2.1. Characterization of U6 promoter in fugu = 58
2.2. Construction of siRNA expression vector targeting EGFP = 58
2.3. Cell culture and transfection = 60
2.4. EGFP knockdown assay = 61
2.5. Northern blot analysis = 61
3. Results = 64
3.1. Characterization of fugu U6 promoter = 64
3.2. Detection of processed siRNA expressed by fugu U6 promoter = 66
3.3. Knockdown of EGFP gene expression = 68
4. Discussion = 71
Chapter Ⅳ. Construction of short interfering RNA (siRNA) expression vector driven by modified rock bream β-actin promoter = 74
1. Introduction = 75
2. Materials and methods = 78
2.1. Determination of the transcription start site of β-actin promoter = 78
2.2. Construction of EGFP expression vector driven by modified β-actin promoter = 78
2.3. Construction of shRNA expression vector targeting EGFP = 80
2.4. Cell culture and transfection = 81
2.5. Quantification of EGFP expression level by fluoremeter = 82
2.6. Quantification of EGFP expression level by semi-quantitative RT PCR = 83
3. Result = 87
3.1 Sequence analysis and determination of TSS of rock bream β-actin gene = 87
3.2 Transcription efficiency of modified β-actin promoter = 89
3.3 Knock down effect of shRNA expression vector driven by modified β-actin promoter = 92
4. Discussion = 95
Chapter Ⅴ. Inhibition of rock bream iridovirus replication in vitro using siRNA expression vector = 99
1. Introduction = 100
2. Materials and methods = 102
2.1. Cells and Virus = 102
2.2. Vector construction = 102
2.3. Transfection and virus challenge = 103
2.4. Growth curve analysis = 104
2.5. Semi-quantitative RT-PCR analysis = 104
3. Result = 107
3.1. CPE observed after inoculation of RBIV = 107
3.2. Growth curve analysis = 109
3.3 Semi-quantitative analysis of viral mRNA expression = 111
4. Discussion = 113
Chapter Ⅵ. Characterization of double-stranded RNA specific ribonuclease encoded by ORF083 of rock bream iridovirus = 117
1. Introduction = 118
2. Materials and methods = 120
2.1. Preparation of recombinant RBVI RNase Ⅲ = 120
2.2. Temporal transcription analysis of RBIV RNase Ⅲ = 121
2.3. Preparation of RNA substrate and cleavage assay = 122
3. Results = 124
3.1. Amino acids sequence of RBIV RNase Ⅲ = 124
3.2. Temporal transcription analysis of RBIV RNase Ⅲ = 125
3.3. Enzyme activity of RBIV RNase Ⅲ = 126
3.4. Effects of NaCl and metal ions concentrations on the RNase Ⅲ activity = 130
4. Discussion = 133
Conclusion = 135
요약 = 136
Acknowledgment = 138
Reference = 140
- Degree
- Doctor
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