720 research outputs found

    Recent progress on technologies and applications of transgenic poultry

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    Manipulation of the poultry genome has the potential to improve poultry production and offer a powerful bioreactor for the production of pharmaceutical and industrial proteins in eggs. However, before realizingthis, the methods for producing transgenic poultry must become routine. The direct modification of an embryo with DNA or viral vectors is possible, but this approach does not have the ability to make locus-specific modifications to the genome. To overcome this problem, several cell-based protocols, which use mainly blastodermal cells (BDCs), embryonic stem cells (ESCs), primordial germ cells (PGCs) and spermatogonial stem cells (SSCs) have been developed to generate transgenic chickens. At present, the complete system for isolating, expanding, transfecting, selecting and re-expanding embryonic stemcell cultures and the subsequent production of high-grade somatic chimeras has been reported, but germline chimeras with transgenic progeny have not yet been achieved. Although the use of viralsystems can achieve highly efficient gene transfer, the potential safety issues may limit their practical application. Among the many possible permutations and combinations of target cell and gene transfermethods described in this review, targeting SSCs in vitro using non-viral-based gene transfer and re-injecting them to cock testis is the most efficient and cost-effective strategy to produce transgenicpoultry

    ์กฐ๋ฅ˜ ์ƒ์‹์„  ์ค„๊ธฐ์„ธํฌ ๋ฐ ์ƒ๋ฌผ ๋ฐ˜์‘๊ธฐ ์‹œ์Šคํ…œ ์‘์šฉ ์—ฐ๊ตฌ

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    ํ•™์œ„๋…ผ๋ฌธ (๋ฐ•์‚ฌ)-- ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› : ๋†์—…์ƒ๋ช…๊ณผํ•™๋Œ€ํ•™ ๋†์ƒ๋ช…๊ณตํ•™๋ถ€(๋ฐ”์ด์˜ค๋ชจ๋“ˆ๋ ˆ์ด์…˜์ „๊ณต), 2018. 2. ํ•œ์žฌ์šฉ.Avian species have become valuable models for biotechnological purposes including production of functional proteins, disease resistance model, and industrial traits as well as developmental studies of vertebrates. To produce genome modified aves, germline-competent stem cells will be required to achieve robust, expedite, and precise genetic modification. Therefore, here, we demonstrated the derivation and manipulation of germline-competent stem cells for various purposes. Firstly, we attempted the derivation of induced pluripotent stem cells-like cells (iPSLCs) from avian feather follicle cells (FFCs). The induced pluripotent stem cell (iPSC) as a novel class of pluripotent stem cells contribute to all three germ layers including germ line. Therefore, we attempted the derivation of induced pluripotent stem cells-like cells (iPSLCs) from avian feather follicle cells (FFCs) as a novel approach to stem cell production. The FFC-iPSLCs can proliferate with the pluripotent property and differentiate into all three germ layers in vitro. This experimental strategy should be useful for conservation and restoration of endangered or high-value avian species without sacrificing embryos. The primordial germ cell (PGC) is the most well established germline-competent stem cells in avian species especially in chicken. In second part, we generated the production of anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B lymphoma and B lymphoblast cells. This experiment well represent as an efficient system for producing anti-cancer therapeutic antibodies from transgenic chicken with consistent expression and highly enhanced Fc effector functions. Meanwhile, to produce efficient genome modification in quail PGCs, we optimize the transfection and drug-selection system into quail genome of primary PGCs. Applying the CRISPR/Cas9 system on quail genome of primary PGCs using electroporation and puromycin drug-selection system, we confirmed that the efficient genome editing ratio compared to non-selected cells. Apart from the PGCs, the spermatogonial stem cells (SSCs) also regarded a reliable way to produce genome-edited animals. In terms of adopting genome modification techniques on quail genome, we produced the germline chimeric quail using testicular cells (TCs) and SSCs transplanted into busulfan-treated recipient testis. The transplantation of male germ cells including spermatogonia and SSCs is an efficient method to study spermatogenesis, control male fertility and transgenesis. Compared with the embryo-mediated method, this strategy is simple and leads to rapid generation of quail germline chimeras. This will lead to production of transgenic models using adult germ cells and, through the production of germline chimeras, also help in efforts to conserve avian species. In conclusion, these experimental strategies for derivation of avian stem cells, manipulation of them and its application using genome editing tools can be used to efficient way for produce birds of economic traits or non-sacrificible avian conservation system.CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. LITERATURE REVIEW 5 1. Induced pluripotent stem cells (iPSCs) for avian conservation 6 1.1. Pluripotent stem cells 6 1.2. iPSCs as a novel pluripotent stem cells 7 1.3. iPSCs for species conservation 8 1.4. A strategy for avian conservation using iPSCs 9 2. The primordial germ cells (PGCs) in avian biotechnology 10 2.1. Derivation and cultivation of PGCs in chicken 11 2.2. PGC-mediated transgenic technology in chicken 12 2.3. PGC-mediated genome editing technology in chicken 13 2.4. Characteristics and purification of quail PGCs 14 2.5. PGC-mediated transgenesis in quail 15 3. Production of recombinant proteins in therapeutic use 16 3.1. Therapeutic recombinant proteins 16 3.2. Bioreactor systems for recombinant protein production 17 3.3. Transgenic avian bioreactor system for production of recombinant proteins 19 4. Transplantation of spermatogonial stem cells (SSCs) for avian transgenesis 23 4.1. Characteristics of SSCs 23 4.2. Transplantation of SSCs and transgenesis in animals 24 4.3. Application of SSCs in avian species 26 CHAPTER 3. INDUCTION OF PLURIPOTENT STEM CELLS-LIKE CELLS FROM CHICKEN FEATHER FOLLICLE CELLS 27 1. Abstract 28 2. Introduction 29 3. Materials and methods . 31 4. Results 37 5. Discussion 47 CHAPTER 4. EVALUATION OF GERMLINE TRANSGENIC CHICKEN SYSTEM AS AN EFFICIENT BIOREACTOR FOR THE PRODUCTION OF THERAPEUTIC ANTIBODY 50 1. Abstract 51 2. Introduction 52 3. Materials and methods 56 4. Results 65 5. Discussion 89 CHAPTER 5. PRODUCTION OF GERMLINE CHIMERIC QUAILS FOLLOWING SPERMATOGONIAL CELL TRANSPLANTATION IN BUSULFAN TREATED TESTIS 95 1. Abstract 96 2. Introduction 97 3. Materials and methods 99 4. Results 104 5. Discussion 114 CHAPTER 6. TARGETED GENE DELETION AND INSERTION ON QUAIL GENOME BY PROGRAMMABLE GENOME EDITING IN PRIMARY PRIMORDIAL GERM CELLS 118 1. Abstract 119 2. Introduction 121 3. Materials and methods 123 4. Results 128 5. Discussion 140 CHAPTER 7. GENERAL DISCUSSION 144 REFERENCES 148 SUMMARY IN KOREAN 190Docto

    Securing poultry production from the ever-present Eimeria challenge

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    ๋ฉ”์ถ”๋ฆฌ์˜ ์œ ์ „์ž ํŽธ์ง‘์„ ์œ„ํ•œ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ์˜ ์‘์šฉ ๋ฐฉ๋ฒ•์— ๋Œ€ํ•œ ์—ฐ๊ตฌ

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    ํ•™์œ„๋…ผ๋ฌธ(์„์‚ฌ)--์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› :๋†์—…์ƒ๋ช…๊ณผํ•™๋Œ€ํ•™ ๋™๋ฌผ์ž์›๊ณผํ•™๊ณผ,2019. 8. ํ•œ์žฌ์šฉ.์กฐ๋ฅ˜๋Š” ๋ชจ๋ธ ๋™๋ฌผ๋กœ์จ ๋ฐœ์ƒํ•™ ์—ฐ๊ตฌ, ์งˆ๋ณ‘ ์ €ํ•ญ์„ฑ ๊ทธ๋ฆฌ๊ณ  ์ƒ์ฒด ๋ฐ˜์‘๊ธฐ ๋ชจ๋ธ ๋“ฑ ๋‹ค์–‘ํ•œ ์‘์šฉ ๊ฐ€๋Šฅ์„ฑ์„ ๊ฐ–๊ณ  ์žˆ๊ณ , ์ด๋Ÿฌํ•œ ๊ฐ€๋Šฅ์„ฑ ๋•Œ๋ฌธ์— ์˜ค๋ž˜์ „๋ถ€ํ„ฐ ์—ฐ๊ตฌ๋˜์–ด์™”๋‹ค. ์ด๋Ÿฌํ•œ ์ƒํ™ฉ์—์„œ ์ƒ์‹์„  ํ‚ค๋ฉ”๋ผ์™€ ํ˜•์งˆ์ „ํ™˜ ์กฐ๋ฅ˜๋ฅผ ์ƒ์‚ฐํ•˜๋Š” ๊ฒƒ์€ ๋งค์šฐ ๊ฐ€์น˜ ์žˆ๋Š” ์—ฐ๊ตฌ์˜€๊ณ , ์กฐ๋ฅ˜์—์„œ ์ƒ์‹์„  ํ‚ค๋ฉ”๋ผ์™€ ํ˜•์งˆ์ „ํ™˜์„ ์œ ๋„ํ•˜๋ ค๋Š” ์ผ๋ จ์˜ ์—ฐ๊ตฌ๋“ค์ด ํ™œ๋ฐœํ•˜๊ฒŒ ๋ณด๊ณ ๋˜์—ˆ๋‹ค. ํŠนํžˆ, ๋‹ญ์˜ ์ƒ๋ฆฌ์ ์ธ ํŠน์„ฑ๊ณผ ๋งค๋…„ ์ˆ˜๋ฐฑ ๊ฐœ ์ด์ƒ์˜ ์•Œ์„ ์‚ฐ๋ž€ํ•œ๋‹ค๋Š” ์ƒ์‹ ํŠน์„ฑ ๋•๋ถ„์— ์กฐ๋ฅ˜ ํ˜•์งˆ์ „ํ™˜ ์—ฐ๊ตฌ์—์„œ ๊ฐ€์žฅ ์ค‘์ ์ ์œผ๋กœ ์—ฐ๊ตฌ๋˜์—ˆ๋‹ค. ํŠนํžˆ, ์กฐ๋ฅ˜ ํ˜•์งˆ์ „ํ™˜ ์—ฐ๊ตฌ์˜ ์ค‘์‹ฌ์—์„œ, ๋‹ญ์˜ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ (germline competent stem cell) ์˜ ํ•œ ์ข…๋ฅ˜์ธ, ์›์‹œ์ƒ์‹์„ธํฌ (primordial germ cell) ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ๊ฐ€ ํ™œ๋ฐœํ–ˆ์œผ๋ฉฐ, ์ฒด์™ธ์—์„œ ์žฅ๊ธฐ ๋ฐฐ์–‘ํ•˜๋Š”๋ฐ ์„ฑ๊ณตํ•˜์˜€๋‹ค. ๋‹ญ์„ ์ œ์™ธํ•œ ๋‹ค๋ฅธ ์กฐ๋ฅ˜์ข…์—์„œ๋„ ์›์‹œ์ƒ์‹์„ธํฌ๋ฅผ ๋ถ„๋ฆฌํ•˜๊ณ  ๋ฐฐ์–‘ํ•˜๊ธฐ ์œ„ํ•œ ์—ฐ๊ตฌ๊ฐ€ ์žˆ์—ˆ์ง€๋งŒ, ํ™•๋ณดํ•  ์ˆ˜ ์žˆ๋Š” ์„ธํฌ์˜ ์ˆ˜๊ฐ€ ๋งค์šฐ ์ ๊ณ , ์žฅ๊ธฐ๊ฐ„์˜ ์ฒด์™ธ ๋ฐฐ์–‘๋„ ๋ถˆ๊ฐ€๋Šฅํ•œ ์ˆ˜์ค€์ด์—ˆ๋‹ค. ๋”ฐ๋ผ์„œ ์ƒ์‹์„  ํ‚ค๋ฉ”๋ผ ๋˜๋Š” ํ˜•์งˆ์ „ํ™˜ ์กฐ๋ฅ˜๋ฅผ ์ƒ์‚ฐํ•˜๊ธฐ ์œ„ํ•ด์„œ ์›์‹œ์ƒ์‹์„ธํฌ๋ฅผ ๋Œ€์ฒดํ•  ์ˆ˜ ์žˆ๋Š” ๋‹ค๋ฅธ ์ข…๋ฅ˜์˜ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ๊ฐ€ ํ•„์š”ํ•˜์˜€๊ณ , ๋น„๊ต์  ํฌ์œ ๋™๋ฌผ์—์„œ ๋งŽ์ด ์—ฐ๊ตฌ๋œ ์„ฑ์ฒด ์ค„๊ธฐ์„ธํฌ์˜ ํ•œ ์ข…๋ฅ˜์ธ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ๊ฐ€ ์กฐ๋ฅ˜์—์„œ๋„ ์š”๊ตฌ๋˜์—ˆ๋‹ค. ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” ๋ฐ€๋„ ๊ตฌ๋ฐฐ ์›์‹ฌ๋ถ„๋ฆฌ ๊ธฐ๋ฒ•์„ ์ด์šฉํ•˜์—ฌ ๋ฉ”์ถ”๋ฆฌ์˜ ์ •์†Œ์„ธํฌ๋กœ๋ถ€ํ„ฐ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ๋ฅผ ๋†์ถ•์‹œ์ผฐ๊ณ , ๋†์ถ•๋œ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ๋ฅผ ๋ฉ”์ถ”๋ฆฌ์˜ ์ •์†Œ์— ์ˆ˜์ˆ ์  ๊ธฐ๋ฒ•์„ ํ†ตํ•ด ์ฃผ์ž…ํ•จ์œผ๋กœ์จ ์ƒ์‹์„  ํ‚ค๋ฉ”๋ผ๋ฅผ ํšจ๊ณผ์ ์œผ๋กœ ์ƒ์‚ฐํ•  ์ˆ˜ ์žˆ์—ˆ๋‹ค. ๋˜ํ•œ, ๋ฉ”์ถ”๋ฆฌ์˜ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์— ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ์œ ์ „์ž ํ‘œ์  ํŽธ์ง‘ ๊ธฐ์ˆ  (programmable genome editing platform) ์˜ ์ผ์ข…์ธ CRISPR/Cas9 system ์„ ๋„์ž…ํ•˜์˜€๊ณ , ํšจ๊ณผ์ ์œผ๋กœ ์ƒ์‹์„  ์ค„๊ธฐ์„ธํฌ ๋‚ด์—์„œ ํšจ๊ณผ์ ์œผ๋กœ ์œ ์ „์ž ๋ณ€ํ˜•์„ ์œ ๋„ํ•˜์˜€๋‹ค. ์ฒซ ๋ฒˆ์งธ ์—ฐ๊ตฌ์—์„œ๋Š” ๋ฉ”์ถ”๋ฆฌ ์ •์†Œ์ค„๊ธฐ์„ธํฌ๋ฅผ Ficoll-Paque PLUS (Ficoll), Percoll, sucrose ์šฉ์•ก์—์„œ ๋ฐ€๋„ ๊ตฌ๋ฐฐ ์›์‹ฌ๋ถ„๋ฆฌ๋ฅผ ์‚ฌ์šฉํ•˜์—ฌ ๋ถ„๋ฆฌํ•˜์˜€๊ณ , qRT-PCR์„ ํ†ตํ•ด์„œ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ ํŠน์ด์  ์œ ์ „์ž (GFRA1, ITGA6, and ITGB1) ์™€ ์ „๋ถ„ํ™”๋Šฅ ๊ด€๋ จ ์œ ์ „์ž (NANOG and POUV) ์˜ ๋ฐœํ˜„์„ ์ •๋Ÿ‰ ํ•˜์˜€๋‹ค. ํฅ๋ฏธ๋กญ๊ฒŒ๋„, ์„ธ ๊ฐ€์ง€ ์‹คํ—˜๊ตฐ ๋ชจ๋‘ ์ƒ์ธต์— ๋ถ„๋ฆฌ๋œ ์„ธํฌ์—์„œ ๋†’์€ ์œ ์ „์ž ๋ฐœํ˜„ ์–‘์ƒ์„ ๋ณด์˜€์œผ๋ฉฐ, Ficoll์— ์˜ํ•ด ๋ถ„๋ฆฌ๋œ ์ƒ์ธต ์„ธํฌ์—์„œ ๊ฐ€์žฅ ๋†’์€ ๋ฐœํ˜„์„ ํ™•์ธํ•˜์˜€๋‹ค. ์ด์–ด์„œ RNA probe hybridzation๊ณผ ํˆฌ๊ณผ์ „์žํ˜„๋ฏธ๊ฒฝ์„ ํ†ตํ•ด Ficoll ์ƒ์ธต ์„ธํฌ์— ์ •์†Œ ์ค„๊ธฐ์„ธํฌ๊ฐ€ ๋†์ถ•๋˜์–ด์žˆ์Œ์„ ํ™•์ธํ•˜์˜€๋‹ค. ์ƒ์‹์„  ์ „์ด (germline transmission) ๋Šฅ๋ ฅ์„ ๊ฒ€์ฆํ•˜๊ธฐ ์œ„ํ•ด ๋†์ถ•๋œ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ๋ฅผ buslufan ์ฒ˜๋ฆฌํ•œ ๋ฉ”์ถ”๋ฆฌ์˜ ์ •์†Œ์— ์ˆ˜์ˆ ์  ๊ธฐ๋ฒ•์œผ๋กœ ์ฃผ์ž…ํ•˜์˜€๊ณ , ๊ฒ€์ • ๊ต๋ฐฐ๋ฅผ ํ†ตํ•ด ์ƒ์‹์„  ์ „์ด ํšจ์œจ์„ ์ธก์ •ํ•˜์˜€๋‹ค. ๊ฒฐ๊ณผ์ ์œผ๋กœ ์ •์†Œ์„ธํฌ๋ฅผ ์ฃผ์ž…ํ•œ ๋Œ€์กฐ๊ตฐ (1.4 ยฑ 1.4 %) ์— ๋น„๊ตํ•˜์—ฌ ์ •์†Œ์ค„๊ธฐ์„ธํฌ๋ฅผ ๋†์ถ• ์‹œํ‚จ ํ›„ ์ฃผ์ž…ํ•œ ์‹คํ—˜๊ตฐ (8.4 ยฑ 1.7 %)์—์„œ ์•ฝ 6๋ฐฐ ๋†’์€ ์ƒ์‹์„  ์ „์ด ํšจ์œจ์„ ํ™•์ธํ•˜์˜€๋‹ค. ๋‘ ๋ฒˆ์งธ ์—ฐ๊ตฌ์—์„œ๋Š”, ๋ถ„๋ฆฌ๋œ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์˜ ์œ ์ „์ž ์ ์ค‘ ํŽธ์ง‘์„ ์œ ๋„ํ•˜๊ธฐ ์œ„ํ•ดCRISPR/Cas9 system์„ ์ฒด์™ธ์—์„œ ๋„์ž…ํ•˜์˜€๋‹ค. ๋จผ์ €, ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค์˜ ์™ธ๋ž˜ ์œ ์ „์ž ์ „๋‹ฌ ํšจ๊ณผ๋ฅผ ๋ฉ”์ถ”๋ฆฌ์˜ ๋ฐฐ์•„ ์ƒ์‹์„  ์„ธํฌ์™€ ๋†์ถ•๋œ ์ •์†Œ์ค„๊ธฐ์„ธํฌ์—์„œ ๊ฒ€์ฆํ•˜์˜€๋‹ค. ๋˜ํ•œ ์„ธํฌ์™€ ๋ฐ”์ด๋Ÿฌ์Šค ์‚ฌ์ด์˜ ๊ฒฐํ•ฉ๋ ฅ์„ ์ฆ์ง„์‹œํ‚ค๊ธฐ ์œ„ํ•ด ์ค‘ํ•ฉ์ฒด์˜ ์ผ์ข…์ธ poly-L-lysine ์„ ์ฒจ๊ฐ€ํ•˜์—ฌ ์ „๋‹ฌ ํšจ๊ณผ๋ฅผ ์ตœ์ ํ™”์‹œ์ผฐ๋‹ค. ์ด๋ ‡๊ฒŒ ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ํ†ตํ•ด GFP ์œ ์ „์ž๊ฐ€ ๋„์ž…๋œ ์›์‹œ์ƒ์‹์„ธํฌ๋ฅผ ํ‘œ๋ฉด ํŠน์ด์  ๋งˆ์ปค๋กœ ๋ถ„๋ฆฌํ•˜๊ณ , ์ •์†Œ ์ค„๊ธฐ์„ธํฌ์™€ ํ•จ๊ป˜ ์ƒ์‹์„ธํฌ ํŠน์ด์  ์œ ์ „์ž (VASA, DAZL), ์ „๋ถ„ํ™”๋Šฅ ๊ด€๋ จ ์œ ์ „์ž (NANOG, POUV), ์ •์†Œ์ค„๊ธฐ์„ธํฌ ํŠน์ด์  ์œ ์ „์ž (GFRA1, ITGA6, ITGB1) ์˜ ๋ฐœํ˜„์„ ๊ฒ€์ฆํ•˜์˜€๋‹ค. ๋˜ํ•œ ๋ฉด์—ญ ์„ธํฌ ํ™”ํ•™ ๊ธฐ๋ฒ•์„ ํ†ตํ•ด DAZL์œ ์ „์ž๋ฅผ ๋ฐœํ˜„ํ•˜๋Š” ์„ธํฌ์—์„œ ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ํ†ตํ•ด ๋“ค์–ด์˜จ GFP ์œ ์ „์ž๊ฐ€ ๋ฐœํ˜„๋˜๋Š” ๊ฒƒ์„ ํ™•์ธํ•˜์˜€๋‹ค. ๋งˆ์ง€๋ง‰์œผ๋กœ, CRISPR/Cas9 system์„ ์ „๋‹ฌํ•˜๊ธฐ ์œ„ํ•œ ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ์ œ์ž‘ํ•˜์˜€๊ณ , ๋ฉ”์ถ”๋ฆฌ์™€ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์—์„œ Transferrin ๊ณผ Hoxb13 ์œ ์ „์ž๋ฅผ ์ ์ค‘ํ•˜์—ฌ ๋ณ€ํ˜•์„ ์œ ๋„ํ•˜์˜€๋‹ค. T7E1 ๋ถ„์„๊ณผ ์—ผ๊ธฐ์„œ์—ด ํ™•์ธ์„ ํ†ตํ•ด ์ ์ค‘๋ถ€์œ„์—์„œ ์—ผ๊ธฐ์„œ์—ด ๋ณ€ํ˜•์ด ์ผ์–ด๋‚œ ๊ฒƒ์„ ํ™•์ธํ•˜์˜€๊ณ , ๋ฉ”์ถ”๋ฆฌ์˜ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์—์„œ ์•ฝ 33.3 % ์˜ ํšจ์œจ๋กœ ์œ ์ „์ž ํŽธ์ง‘์ด ์ผ์–ด๋‚จ์„ ํ™•์ธํ–ˆ๋‹ค. ๊ฐ™์€ ๋ฐฉ๋ฒ•์„ ๋‹ญ์˜ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์—๋„ ์ ์šฉํ•˜์˜€์ง€๋งŒ, ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค์˜ ์ „๋‹ฌ ํšจ์œจ์ด ๋งค์šฐ ๋‚ฎ์•˜๋‹ค. ๊ฒฐ๊ณผ์ ์œผ๋กœ, ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” Ficoll์— ์˜ํ•œ ๋ฐ€๋„ ๊ตฌ๋ฐฐ ์›์‹ฌ๋ถ„๋ฆฌ๋ฅผ ํ†ตํ•ด์„œ ๋ฉ”์ถ”๋ฆฌ์˜ ์ •์†Œ ์ค„๊ธฐ์„ธํฌ๋ฅผ ๋†์ถ•ํ•˜๋Š”๋ฐ ์„ฑ๊ณตํ•˜์˜€์œผ๋ฉฐ, ๋†์ถ•๋œ ์ •์†Œ์ค„๊ธฐ์„ธํฌ์˜ ์ •์†Œ์„ธํฌ๋กœ์˜ ์ด์‹์„ ํ†ตํ•ด ์ƒ์‹์„  ์ „์ด ๋Šฅ๋ ฅ์ด ํ–ฅ์ƒ๋จ์„ ๊ฒ€์ฆํ•˜์˜€๋‹ค. ๋˜ํ•œ ์ฒด์™ธ์—์„œ ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ํ†ตํ•ด CRISPR/Cas9 system์„ ๋ฉ”์ถ”๋ฆฌ ์ƒ์‹์„  ์ „์ด ์ค„๊ธฐ์„ธํฌ์— ํšจ๊ณผ์ ์œผ๋กœ ์ „๋‹ฌํ•˜์˜€๊ณ , ์ ์ค‘ํ•˜๊ณ ์žํ•œ ๋ถ€์œ„์—์„œ ์œ ์ „์ž ์—ผ๊ธฐ์„œ์—ด์˜ ๋ณ€ํ˜•์„ ํ™•์ธํ•˜์˜€๋‹ค. ๋ณธ ์—ฐ๊ตฌ์—์„œ ๋ฐํžŒ ์ •์†Œ์ค„๊ธฐ์„ธํฌ ๋†์ถ• ๊ธฐ๋ฒ•๊ณผ ์•„๋ฐ๋…ธ๋ฐ”์ด๋Ÿฌ์Šค๋ฅผ ํ†ตํ•œ ์œ ์ „์ž ์กฐ์ ˆ ๊ธฐ๋ฒ•์€ ์—ฌ๋Ÿฌ ๋ฉธ์ข…์œ„๊ธฐ ์กฐ๋ฅ˜ ์ข…์˜ ๋ณต์›๊ณผ ์œ ์ „์ž ์กฐ์ ˆ ์กฐ๋ฅ˜์˜ ์ƒ์‚ฐ ์—ฐ๊ตฌ์— ๋Œ€ํ•œ ๊ธฐ์ดˆ ์ž๋ฃŒ๋กœ์จ ๊ธฐ์—ฌ ํ•  ์ˆ˜ ์žˆ๋‹ค.Avian species have been considered as one of the most valuable animal models for various applications including developmental biology, disease resistance model and bioreactor. In this circumstance, efforts for generating germline chimeric and transgenic birds have been studied for a long time by a number of investigators. In particular, chicken has been focused as a useful bioreactor model with their high egg production rate. Even, primordial germ cell (PGC), which is one of germline competent stem cells, have been well investigated while long-term in vitro culture has been established in chicken. However, it was hard to apply chicken primordial germ cell culture system to other avian species due to lack of cell sources and short in vitro culture duration. Thus, it is necessary to develop alternative germline competent stem cell mediated germline chimeric and transgenic bird generation in other avian species. In this study, we established simple and practical density gradient centrifugation mediated spermatogonial stem cell (SSC) enrichment method, and verified feasibility and enhancement of enriched spermatogonial stem cells germline transmission efficiency in quail. Furthermore, we induced in vitro genome modification in isolated and enriched quail germline competent stem cells with adenoviral vector mediated CRISPR/Cas9 genome editing tool. From the first study, we enriched quail SSC by density gradient centrifugation methods with Ficoll-Paque PLUS (Ficoll), Percoll and sucrose solutions. To evaluate enrichment of each fractions, expression levels of SSC-specific genes (GFRA1, ITGA6, and ITGB1) and pluripotency genes (NANOG and POUV) were examined by qRT-PCR. Interestingly, cells from upper fractions in most of density gradients showed significantly higher gene expressions. In addition, qRT-PCR results revealed that cells from upper fractions in Ficoll density gradient showed the highest SSC-specific and pluripotency marker expression. Then we confirmed SSC enriched fractions by RNA hybridization and TEM image. Subsequently, SSC enriched fractions were transplanted into busulfan treated quail testis, and PKH-26 labeled donor cells were detected from the testicular tubules. We performed testcross analysis for verifying germline transmission efficiency. As a results, SSC enriched fraction transplanted quail produced donor-derived sperm and progeny, and its efficiency (8.4 ยฑ 1.7 %) showed significantly 6 times higher than that of whole testicular cells transplanted group (1.4 ยฑ 1.4 %). Finally, we introduced CRISPR/Cas9 system into quail germline competent stem cells for inducing in vitro genomic DNA modification. We transduced embryonic gonadal cells and enriched SSC by adenoviral and adeno-associated viral vectors, and only adenoviral vector showed positive signals in all quail cells. Then we optimized adenoviral transduction with poly-L-lysine adding, and 1 ยตg/mL concentration of poly-L-lysine mostly optimized adenoviral transduction in all quail cells. For characterizing adenoviral vector transduced cells, we identified germ cell specific (VASA, DAZL), pluripotency (NANOG, POUV) and germline stem cell specific (GFRA1, ITGA6, ITGB1) markers from quail PGC, SSC and QM7 cell line. Finally, we constructed adenoviral vector delivering CRISPR/Cas9 targeting for Transferrin and Hoxb13 gene, and induced genome modification in quail germline competent stem cells. T7E1 assay and sequencing analysis revealed that CRISPR/Cas9 delivery with adenoviral vector induced about 33.3 % of mutation at genomic DNA of quail germline competent stem cells. However, there was no positive signal with adenoviral and adeno-associated viral vectors in chicken germline competent stem cells. Collectively, these results suggested that Ficoll density gradient solution can be used as a simple and practical method for SSC enrichment, and this method could be applied for bird conservation, restoration and even transgenic quail researches. In addition, adenoviral vector mediated CRIPSR/Cas9 delivering is efficient in quail germline competent stem cells, but not in chicken germline competent stem cells. Thus, we can expect to apply adenoviral vector with CRISPR/Cas9 system via in vitro even in vivo approaches for generating targeted genome edited quail.CHAPTER 1. GENERAL INTRODUCTION 1 CHAPTER 2. LITERATURE REVEIW 6 1. Quail as a research model bird 7 1.1. Quail as a bird conservation model 7 1.2. Quail as a developmental biology model 8 2. Germline competent stem cell in avian biotechnology 9 2.1. Primordial germ cell (PGC) 10 2.2. Spermatogonial stem cell (SSC) 11 2.3. Transplantation of SSC in animals 14 2.4. Applications of SSC in avian species 16 3. Programmable genome editing technology 17 3.1. Zinc finger nuclease (ZFN) 17 3.2. Transcription activator-like effector nuclease (TALEN) 18 3.3. Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associate protein 9 (CRISPR/Cas9) 18 3.4. Programmable genome editing in avian species 19 4. Adenoviral vector 20 4.1. Adenoviral vector as a tool for gene therapy 21 4.2. Adenoviral vector mediated transgenesis in animals 22 4.3. Adenoviral vector mediated CRISPR/Cas9 delivery in animals 23 CHAPTER 3. ENRICHMENT OF SPERMATOGONIAL STEM CELL BY DENSITY GRADIENT CENTRIFUGATION AND ITS TESTICULAR TRANSPLANTATION FOR GERMLINE TRANSMISSION 24 1. Introduction 25 2. Materials and methods 29 3. Results 35 4. Discussion 45 CHAPTER 4. IN VITRO GENOME MODIFICATION OF QUAIL GERMLINE COMPETENT STEM CELLS BY ADENOVIRAL VECTOR MEDIATED CRISPR/CAS9 DELIVERY 51 1. Introduction 52 2. Materials and methods 56 3. Results 62 4. Discussion 75 CHAPTER 5. GENERAL DISCUSSION 80 REFERENCES 85 SUMMARY IN KOREAN 107Maste

    Role of CSF1/CSF1R signalling in avian macrophage biology

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    The mononuclear phagocyte system (MPS), which is a heterogenous family of functionally related cells, includes myeloid progenitors, blood monocytes, resident tissue macrophages, bone osteoclasts and conventional dendritic cells. In mammals, macrophage colony stimulating factor (M-CSF or CSF1) promote differentiation, proliferation and survival of myeloid progenitor cells into mononuclear phagocyte lineage cells by binding and signalling activity through a surface receptor (CSF1R). Interleukin-34 or IL34 is alternative growth factor which also signals via CSF1R. CSF1, IL34 and the shared receptor CSF1R was shown to be conserved in birds, but their functions have not been studied in detail. The primary aim of this project is to study the role of CSF1R signalling in avian macrophage biology using three different approaches. The first approach involved the identification of chicken CSF1R specific kinase inhibitors, from a set of candidate mammalian CSF1R. Candidate CSF1R inhibitors were screened based on cell viability assay using IL-3 dependent pro B cell line Ba/F3 ectopically expressing chicken CSF1R and chicken bone marrow-derived macrophages (BMDM). To support these studies, biologically active, endotoxin-free recombinant chicken CSF1 protein was produced and refolded from inclusion bodies using a bacterial system. Out of 10 potential CSF1R inhibitors screened, 6 inhibitors TIA086, TIA02-052, TIA02-054, TIA02-076, KUL01-123 and KUL02- 016 were potent and selective for chicken CSF1R, whilst having no effect on growth in IL-3. Two inhibitors TIA02-054 and TIA02-076 were specific for the chicken CSF1R kinase compared to their actions on human CSF1R expressed in the same cells. The chicken CSF1R kinase inhibitors also effectively blocked CSF1-induced survival of primary BMDMs. BMDM survival was reduced even in the absence of exogenous CSF1 indicating a growth factor independent, autocrine CSF1/CSF1R signalling function in chicken macrophages. The second approach to study CSF1 biology in the development of chicken MPS involved use of a novel neutralising monoclonal antibody to chicken CSF1 (ROS-AV183) that targets and blocks chicken CSF1R signalling activity. In order to test the activity of anti-ChCSF1 mAb on chicken macrophages both in vitro and in vivo, both anti-ChCSF1 mAb and Isotype control mAb reagents were purified from hybridoma culture by affinity chromatography and characterized further for purity, size by SDS PAGE and CSF1R signaling blocking activity by BaF3/ChCSF1R cell viability assay. Anti-ChCSF1 mAb completely inhibited survival of primary chicken macrophages, irrespective of the presence or absence of CSF1, supporting the earlier finding regarding the autocrine CSF1 signalling behaviour of chicken macrophages. To determine the impact of anti-ChCSF1 mAb on postnatal birds in vivo, transgenic CSF1R-eGFP reporter birds were injected with antibody for four consecutive days. Anti-ChCSF1 mAb had no effect on the average growth rate, the relative weight gain or the normal development of hatchling birds. Anti-ChCSF1 mAb had no detectable effect on circulating CSF1 levels on the day of hatch or a week after treatment. Anti-ChCSF1 mAb significantly reduced CSF1R-eGFP transgene positive macrophages in bursa of Fabricius and caecal tonsil tissue, but not in spleen tissue. In bursa of Fabricius tissue, follicle associated epithelium (FAE) cellโ€™s proliferation and survival was altered post treatment. In caecal tonsil anti-ChCSF1 mAb substantially reduced B lymphocytes; this depletion was also evident in the circulation and spleen tissue. Tissue resident MHC-II+ macrophages in spleen were effectively depleted, validating CSF1 dependency of tissue resident macrophages. In liver tissue, anti-ChCSF1 mAb treatment completely ablated Kupffer cell population. In bones anti-ChCSF1 mAb treatment depleted osteoclasts number. MicroCT scan analysis of bone femur architecture revealed significant reduction in the % bone volume and trabecular number, with a corresponding increase in the trabecular separation post anti-ChCSF1 mAb treatment of hatchling birds. In overview, the analysis indicated that CSF1 is required for post-hatch development of the MPS in birds and suggest trophic roles for CSF1-dependent macrophages in B cell development. The third approach involved deletion of CSF1R in the chicken genome using CRISPR Cas9 editing in chicken primordial germ cells (PGCs). Out of the several guide RNAs (gRNAs) designed targeting different regions of CSF1R loci, gRNAs targeting exon 1 and 10 (encoding transmembrane domain of the receptor) were functionally validated for mutation. Guide RNAs targeting exon 1 and transmembrane domain region were effective in mutating receptor CSF1R in cultured PGCs with targeting efficiency of around 35% and 100% respectively. Transplantation of PGCs with biallelic deleted transmembrane domain region of CSF1R into germ cell deficient chicken embryos gave rise to one founder female G0 bird containing edited donor PGCs. Breeding of this chicken upon sexual maturation with transgenic CSF1R-eGFP male established 30 CSF1R heterozygous G1 birds containing CSF1R edited donor PGCs (39% germline efficiency). CSF1R heterozygous G1 birds had no obvious phenotypes compared to wild type hatch mates throughout the development of embryos and in adults. Furthermore, CSF1R homozygous mutant embryos (G2) were generated by breeding CSF1R heterozygous G1 chickens (26% germline efficiency). Analysis of 8-day old CSF1R homozygous mutant embryos revealed deficiency in the expression of CSF1R protein in mononuclear phagocyte population. Hence, there was successful transmission of CSF1R knockout allele in G1 and G2 progeny. Analysis of the phenotype of the homozygous CSF1R mutant birds is ongoing. The novel tools characterized in this project, anti-ChCSF1 antibody, chicken CSF1R kinase domain inhibitors and CSF1R-deficient transgenic chicken line will enable further detailed studies of the role of macrophages in chicken immunity and development

    Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.

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    Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity

    Advances in Plant Molecular Farming

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    Plant molecular farming (PMF) is a new branch of plant biotechnology, where plants are engineered to produce recombinant pharmaceutical and industrial proteins in large quantities. As an emerging subdivision of the biopharmaceutical industry, PMF is still trying to gain comparable social acceptance as the already established production systems that produce these high valued proteins in microbial, yeast, or mammalian expression systems. This article reviews the various cost-effective technologies and strategies, which are being developed to improve yield and quality of the plant-derived pharmaceuticals, thereby making plantbased production system suitable alternatives to the existing systems. It also attempts to overview the different novel plant-derived pharmaceuticals and non-pharmaceutical protein products that are at various stages of clinical development or commercialization. It then discusses the biosafety and regulatory issues, which are crucial (if strictly adhered to) to eliminating potential health and environmental risks, which in turn is necessary to earning favorable public perception, thus ensuring the success of the industr

    Recent Advances in the Application of CRISPR/Cas9 Gene Editing System in Poultry Species

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    CRISPR/Cas9 system genome editing is revolutionizing genetics research in a wide spectrum of animal models in the genetic era. Among these animals, is the poultry species. CRISPR technology is the newest and most advanced gene-editing tool that allows researchers to modify and alter gene functions for transcriptional regulation, gene targeting, epigenetic modification, gene therapy, and drug delivery in the animal genome. The applicability of the CRISPR/Cas9 system in gene editing and modification of genomes in the avian species is still emerging. Up to date, substantial progress in using CRISPR/Cas9 technology has been made in only two poultry species (chicken and quail), with chicken taking the lead. There have been major recent advances in the modification of the avian genome through their germ cell lineages. In the poultry industry, breeders and producers can utilize CRISPR-mediated approaches to enhance the many required genetic variations towards the poultry population that are absent in a given poultry flock. Thus, CRISPR allows the benefit of accessing genetic characteristics that cannot otherwise be used for poultry production. Therefore CRISPR/Cas9 becomes a very powerful and robust tool for editing genes that allow for the introduction or regulation of genetic information in poultry genomes. However, the CRISPR/Cas9 technology has several limitations that need to be addressed to enhance its use in the poultry industry. This review evaluates and provides a summary of recent advances in applying CRISPR/Cas9 gene editing technology in poultry research and explores its potential use in advancing poultry breeding and production with a major focus on chicken and quail. This could aid future advancements in the use of CRISPR technology to improve poultry production
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