외래 단백질이 발현하는 형질전환 닭의 생산

다국어 초록 (Multilingual Abstract)

At present, most available biopharmaceutics are produced from cultured animal cells. However, one of the big problems of this producing systems is high production cost due to the difficulty of purification of pharmaceutics from the complex ingredients of culture medium. As a promising solution for this problem, use of “bioreactors” to address the growing demand for large quantities and increasing number of biopharmaceuticals is of prime strategic relevance to medical advancement. Most recently, chickens have been proposed as a highly efficient animal for the production of pharmaceutical proteins versus mammary gland based bioreactors. Some of the advantages of a chicken bioreactor system over its mammalian counterpart include shorter generation time, lower breeding expense and fecundity of chickens.
Transgenic chicken studies have been performed for this doctorate dissertation. In the fist study, tetracycline-inducible expression system was examined to determine whether it could regulate expression of a foreign protein in a transgenic chicken model. This is because uncontrolled constitutive expression of foreign proteins has been known to cause serious physiological disturbances in transgenic animals. The use of a tetracycline-inducible expression system in transgenic chickens represents a potentially viable approach to two competing endpoints: maximal transgene expression with minimal physiological dysfunctions. I have demonstrated successful inducible expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative, and complete reversion of the induced GFP expression to pre-induction level when the inducer was removed from the diet. In addition, stable germline transmission of the exogenous transgene was confirmed in progeny chickens. In the second study, I have designed recombinant secretory EGFP (EGFPSec) in which the signal peptide sequence of the rat follicle-stimulating hormone (FSH) β-subunit was fused upstream of EGFP. Efficient secretion of the modified EGFP has been found in several cell types. Consequently, the level of the gene expression was able to be easily quantified. Application of our modified EGFP expression cassette will also be very helpful in the study of transgenic livestock intended to use as bioreactors for mass production of pharmaceuticals. The results obtained from this two main studies demonstrate the possible use of chicken as bioreactor producing foreign proteins. In addition, these results also significantly provide basic scientific knowledge in avian reproductive physiology.

목차 (Table of Contents)

• Chapter Ⅰ. 서론과 연구사 1
• 1. 서 론 1
• 2. 이론적 배경 6
• 2.1. Retrovirus 6
• 2.2. Retrovirus vector system 8
• 2.3. Packaging cell and Producing cell 11
• 2.4. Problems of retrovirus vectors in gene transfer 14
• 3. Tetracycline inducible expression system 17
• 3.1. Transgene expression system 17
• 3.2. Tetracycline controllable retrovirus vector system 17
• 4. Transgenic chickens 21
• 4.1. Transgenic animal 21
• 4.2. Production of transgenic chickens 21
• 4.3. Advantages of chickens as bioreactors 27
• 5. Reporter gene 28
• 5.1. Properties of green fluorecent protein 28
• 6. References 30
• Chapter Ⅱ. Production of Transgenic Chickens Expressing a Tetracycline-inducible GFP gene 50
• 1. Abstract 50
• 2. Introduction 51
• 3. Materials and Methods 54
• 3.1. Construction of retrovirus vector and virus production 54
• 3.2. Gene transfer and ex ovo culture of the embryos 57
• 3.3. Genomic DNA analyses 57
• 4. Results 59
• 4.1.Generation of transgenic chickens harboring the GFP gene under a tetracycline-controllable promoter 59
• 4.2. Induction of GFP expression by feeding doxycycline 59
• 4.3. Germline transmission of the transgene 62
• 5. Discussion 64
• 6. References 67
• Chapter Ⅲ. Quantitative Analysis of Tetracycline Inducible Expression of the Green fluorecent Protein Gene in Transgenic Chickens 71
• 1. Abstract 71
• 2. Introduction 72
• 3. Materials and Methods 75
• 3.1. Primary culture of chicken embryonic cells 75
• 3.2. Genomic DNA analysis 75
• 3.3. Quantitative analysis of GFP expression 76
• 3.4. Test of helper virus production 77
• 4. Results and Discussion 79
• 4.1. Breeding of non-mosaic transgenic chickens 79
• 4.2. Induction of GFP expression by feeding of doxycycline 83
• 4.3. Quantitative analysis of doxycycline-induced gene expression 86
• 5. References 90
• Chapter Ⅳ. Modification of Enhanced Green Fluorescent Protein for Secretion Out of Cells 93
• 1. Abstract 93
• 2. Introduction 94
• 3. Materials and Methods 97
• 3.1. Construction of retrovirus vectors 97
• 3.2. Cell culture and virus production 97
• 3.3.Preparation of cell lysate and culture medium sample for fluorescence assay 98
• 3.4. Fluorometric assay 99
• 3.5. Western Blot analysis 99
• 4. Results and Discussion 100
• 4.1. Secretion of EGFP from different cell types 100
• 4.2. Quantification of intra- and extracellular EGFP 104
• 5. Conclusion 110
• 6. References 111