본문 바로가기 주메뉴 바로가기
22대 대한민국 국회 국회정보길라잡이 국회의원검색
회원가입 로그인
HOME

정보검색

소장정보 검색

국회도서관 홈으로 정보검색 소장정보 검색
결과 내 검색 동의어 포함
결과 내 검색 동의어 포함

-

목차보기

표제지

목차

List of abbreviations 10

Abstract 11

Chapter I. 서론과 연구사 13

1. 서론 13

2. 이론적 배경 18

2.1. Retrovirus 18

2.2. Retrovirus vector system 20

2.3. Packaging cell and Virus-producing cell 23

2.4. Problems of retrovirus vector in gene transfer 26

3. Tetracycline inducible expression system 29

3.1. Transgene expression system 29

3.2. Tetracycline controllable retrovirus vector system 29

4. Transgenic chickens 33

4.1. Transgenic animal 33

4.2. Production of transgenic chickens 33

4.3. Advantages of chickens as bioreactors 39

5. Reporter gene 40

5.1. Properties of green fluorecent protein 40

6. References 42

Capter II. Production of Transgenic Chickens Expressing a Tetracycline-inducible GFP gene 62

1. Abstract 62

2. Introduction 63

3. Materials and Methods 66

3.1. Construction of retrovirus vector and virus production 66

3.2. Gene transfer and ex ovo culture of the embryos 69

3.3. Genomic DNA analyses 69

4. Results 71

4.1. Generation of transgenic chickens harboring the GFP gene under a tetracycline-controllable promoter 71

4.2. Induction of GFP expression by feeding doxycycline 71

4.3. Germline transmission of the transgene 74

5. Discussion 76

6. References 79

Chapter III. Quantitative Analysis of Tetracycline Inducible Expression of the Green fluorescent Protein Gene in Transgenic Chickens 83

1. Abstract 83

2. Introduction 84

3. Materials and Methods 87

3.1. Primary culture of chicken embryonic cells 87

3.2. Genomic DNA analysis 87

3.3. Quantitative analysis of GFP expression 88

3.4. Test of helper virus production 89

4. Results and Discussion 91

4.1. Breeding of non-mosaic transgenic chickens 91

4.2. Induction of GFP expression by feeding of doxycycline 95

4.3. Quantitative analysis of doxycycline-induced gene expression 98

5. References 102

Chapter IV. Modification of Enhanced Green Fluorescent Protein for Secretion Out of Cells 105

1. Abstract 105

2. Introduction 106

3. Materials and Methods 109

3.1. Construction of retrovirus vectors 109

3.2. Cell culture and virus production 109

3.3. Preparation of cell lysate and culture medium sample for fluorescence assay 110

3.4. Fluorometric assay 111

3.5. Western Blot Analysis 111

4. Results and Discussion 112

4.1. Secretion of EGFP from different cell types 112

4.2. Quantification of intra- and extracellular EGFP 116

5. Conclusion 122

6. References 123

List of Tables

Table. 1. Hatchability and transgenic efficiency. 72

List of Figures

Chapter I. 8

Fig. 1. The life cycle of a typical retrovirus. Retrovirus infection leads to reverse transcription of viral RNA to proviral DNA, which is integrated into the host genome.... 19

Fig. 2. Structure of MoMLV provirus and its full transcript. 22

Fig. 3. Schematic strategy for the construction of replication defective virus producing cell and introduction of genes into cells. 25

Fig. 4. Diagrams of two controllable gene expression systems. In tet-off system, there are two major elements, specifically, a chimeric tetrachcline-controlled transactivator (tTA) which is a... 31

Fig. 5. Strategy of transgenic chicken production. Construction of recombinant retrovirus vector, production of virus, and the infection of embryos are followed by ex ovo culture, hatching,... 38

Capter II. 8

Fig. 1. Schematic representation of the retrovirus vector constructs. Genes of HygR, EGFP, and rtTA2SM2 in all vectors were expressed under the control of LTR, TREtight, and PGK promoters, respectively....(이미지참조) 68

Fig. 2. Expression of the GFP gene after tetracycline induction. The G0 transgenic chicken (ID number, TGP 22) fed with doxycycline for 2 weeks were illuminated with UV light and photographed head (A),... 73

Fig. 3. Detection of the transgene in G1 transgenic chickens. 75

Fig. 4. Expression of the GFP gene in a G1 transgenic chicken (TGP 22-89) fed doxycycline. The chicken was fed doxycycline for 14 days, and then fed a doxycycline-free diet for 64 days.... 78

Chapter III. 9

Fig. 1. Analysis of G2 transgenic chickens. 93

Fig. 2. Expression of GFP in a G2 transgenic chicken (TGP 22-89-12) fed doxycycline. Doxycycline was supplied in the diet for 14 days, followed by a doxycycline-free diet for 39 days.... 96

Fig. 3. Induction of GFP expression in vitro. Chicken embryonic fibroblast (CEF) cells isolated from a G3 transgenic chicken embryo were treated with doxycycline (1 ㎍/㎖) for 6 days and then cultured in... 97

Fig. 4. Quantitative analyses of tetracycline-inducible expression of GFP. In immunoblot analysis (A-i), 10 ng of purified recombinant GFP (purchased from Clontech) for the positive control or 5 ㎍ of CEF... 100

Chapter IV. 9

Fig. 1. (A) Structure of the LNCGW provirus. LTR, long terminal repeat of Moloney murine sarcoma virus; NeoR neomycin resistance gene; CMVp, human cytomegalovirus promoter; EGFP, enhanced green...(이미지참조) 113

Fig. 2. Detection of green fluorescence in cells, cell lysates and culture media following infection with either the LNCGW or LNCsGW virus. 115

Fig. 3. Fluorometric measurement of EGFP in lysates and culture media from different cells. Green fluorescence was measured as described in the Materials and Methods.... 119

Fig. 4. Western blot analysis of EGFP in lysates and culture media from different cells. Proteins (~10 ㎍) were separated by electrophoresis and transferred onto a PVDF membrane as described in the... 121

초록보기

 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.

추천서가 (다양한 추천 자료를 만나보세요)

권호기사보기

권호기사 목록 테이블로 기사명, 저자명, 페이지, 원문, 기사목차 순으로 되어있습니다.
기사명 저자명 페이지 원문 기사목차