Generation of various transgenic marine medaka (Oryzias dancena) strains harboring different fluorescent transgenic constructs
- Alternative Title
- 다양한 형광발현벡터가 이식된 형질전환 바다송사리 (Oryzias dancena) 개발
- Abstract
- Transgenesis in small aquarium fish models can provide a number of advantageous merits as an experimental platform for various theoretical and practical researches. Marine medaka is one of novel candidates that could be subjected to ornamental application by using a transgenesis of fluorescent protein genes, due to its transparent body color and great osmoregulatory capacity. In this study, three tissue-specific promoters: two promoters derived from a muscle-specific gene encodes a myosin light chain 2 polypeptide (mlc2) and skeletal α-actin were used to express cyan fluorescent protein (CFP) and red fluorescent protein (RFP), respectively; and the another promoter, cytoskeletal β-actin, is a conserved housekeeping protein was also capable of directing endogenous GFP expression in transient transgenic marine medaka embryos and adults. Although transgenic founders exhibited mosaic expression, most of established transgenic germ-lines passed on the fluorescent transgene into their subsequent generations following the Mendelian single gene inheritance pattern. However, transgenic expression particularly in the sense of intensity of fluorescent signals could be largely governed by the kinds of promoters with differential strengths. CFP transgenic fish exhibited distinctive characteristics in terms of fluorescent intensity and hue saturation of the color, while the α-actinRFP displays tissue and developmental specificity in their expression during ontogenesis. On the other hand, the β-actin transgenic expression is the various strain-dependent or sex-related patterns of fluorescent signal during germ-line transmission from parental fish to their offspring. Data from this study could be a useful basis to select both the fluorescent protein genes and promoters for further development of various versions of different fluorescent and ornamental transgenic strains.
- Issued Date
- 2012
- Awarded Date
- 2012. 8
- Type
- Dissertation
- Publisher
- Pukyong National University
- Alternative Author(s)
- 원 탄 부
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 수산생물학과
- Advisor
- Kim Dong Soo
- Table Of Contents
- Abstract i
Table of contents ii
List of tables v
List of figures vi
ACKNOWLEDGEMENTS viii
Chapter 1 - Introduction 1
1. Introduction to transgenic marine medaka Oryzias dancena as a model for fish biology 1
2. Fluorescent transgenic fish as ornamental fish 2
3. Microinjection – a suitable method for transgenesis fish 4
4. The focus of this thesis 5
Chapter 2 - Characterization of cyan fluorescent protein gene (cfp)-transgenic marine medaka strains for ornamental application 6
1. Introduction 6
2. Materials and Methods 10
2.1. Fish maintenance and egg collection 10
2.2. Plasmid preparation for microinjection 10
2.3. Microinjection and identification of positive injected embryos 11
2.4. Breeding of transgenic marine medaka 12
2.5. Characterization of fluorescent phenotypes 12
3. Results and Discussion 14
3.1. Microinjection and stable of CFP-transgenic marine medaka lines 14
3.2. Developmental and ontogenic expression patterns of transgenic CFP signals 18
3.3. Phenotypic characteristics of CFP-transgenic fish under different light sources 21
4. Summary 26
Chapter 3 - Development of stable line and expression of green fluorescent protein in transgenic marine medaka (Oryzias dancena) by using a cytoskeletal β-actin promoter 27
1. Introduction 27
2. Materials and Methods 30
2.1. Preparation of plasmid DNA for microinjection 30
2.2. Microinjection and embryonic development 31
2.3. Germ-line test of transgenic transmission 31
2.4. GFP signals observation 31
3. Results 32
3.1. Transgenic expression of pod β-actinGFP injected embryos and larvae 32
3.2. Frequency of germ-line transmission of injected pod β-actin-GFP sequences 34
3.3. Transmission pattern of green fluorescent transgene from F1 to F2 offspring 36
3.4. Expression of podβ-actinGFP fusion protein to F1 and F2 offspring 39
4. Discussion 45
4.1. Microinjection and frequency of germ-line transmission 45
4.2. Mosaic founders and GFP expression in F1 generation 46
4.3. Contribution for ornamental fish development program 48
5. Summary 50
Chapter 4 - Characteristics of red fluorescent protein harboring the alpha skeletal actin promoter in transgenic marine medaka 51
1. Introduction 51
2. Materials and Methods 53
2.1. Preparation of plasmid DNA and microinjection 53
2.2. Detection of skeletal α-actinRFP transgenic fish 54
2.3. Verification of germ-line transmission 54
3. Results and Discussion 56
3.1. Expression of α-actinRFP in injected embryos 56
3.2. Germ-line transmission pattern of podα-actinRFP in F1 generation 60
3.3. Ontogenic profiling of α-actinRFP transgenic marine medaka 62
4. Summary 69
Chapter 5 - CONCLUSION 70
REFERENCES 71
- Degree
- Master
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