다양한 유전자 조절기술을 이용한 형질전환돼지의 생산

학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2015. 2. 장구.The most ideal way to produce cloned pigs to date is SCNT. Due to comparative simplicity of overexpression than gene KO, lots of target gene overexpressed pigs have been produced while less KO pigs were reported. Recently, diverse genetic scissors (i.e., ZFN, TALEN or RGEN), which rapidly increase the number of KO animals including pigs have been started to highlight. Pigs are considered to be an important large animal model in biomedical research. Due to absence of porcine ESCs and characterized cell lines, development velocities of genetic modification technologies and transgenic pig production are tardy. In this study, to overcome absence of characterized cell lines, hTERT genes overexpressing immortalized cell lines were established and characterized. These cells were further used to apply genetic modification technology (i.e., KO by TALEN). The gene overexpression and KO technologies were respectively applied for genetic modified pig production (i.e., Neurodegenerative disorder pig model). For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the hTERT transfection. After selecting cells with neomycin for two weeks, outgrowing colonized cells were picked up and sub-cultured for expansion. Various analyses were accomplished and characterized for confirming target gene insertion in cells. Immortalized cells were cultured for more than 9 months without changing their doubling time (approximately 24 h) or their diameter (< 20 μm) while control cells became senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to KO the CMAH gene, designed plasmids encoding a TALENs pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation sensitive T7E1 assay, fPCR and sequencing to obtain 3 independent clonal populations of cells that contained biallelic modifications. The GGTA1 gene KO processes was exactly same with that of CMAH KO cell line establishment method. One hTERT overexpressed plus CMAH KO clone was chosen and used for SCNT. Cloned embryos developed to the blastocyst stage. To produce target gene overexpressed and knocked out pigs, PD related genes were selectedSNCA and PRKN. Here, I produced 23 cloned pigs expressing human SNCA via SCNT. Among them, 4 pigs survived (designated PDF4, PDF16, PDF18 and PDF20) and were confirmed as transgenic animals by PCR based analysis. Target proteins were detected in only 2 transgenic pigs (PDF4 and PDF18) by ELISA at 4 months of age. However, SNCA concentrations in blood samples were changed at 1 year of age. PDF4 and PDF20 showed significantly increased levels of SNCA. The SNCA concentration in PDF18 significantly decreased, while that of PDF16 did not change during 1 year after birth. Behavior scoring analysis was carried out in control pigs and all cloned pigs at timed intervals (Control, unchangedPDF18, decreasedPDF4, PDF16 and PDF20, increasedPD like behavioral changes lead score increasing). Similar changes were detected in both ELISA and behavior scoring analyses at timed intervals. PRKN gene was chosen for KO pig production. PRKN specific TALEN pairs were transfected in to pig somatic cells with reporter, 2 to 3 days after transfection GFP signal released cells were chosen for SCNT without specific selection processes. Eight cloned piglets were born and named, PDM1 to 8. In three (PDM3heterozygote, PDM5homozygote, PDM6homozygote) out of eight, PRKN mutation was confirmed. However, these three KO piglets were dead (PDM3unknown reason, PDM5euthanasia because of cleft, PDM6stillbirth). Nevertheless cell lines from those KO pigs except PDM6 were established and will be employed for re-cloned KO piglets. In conclusion, I demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene and the TALEN pairs enabled gene disruptions in these immortalized cells in vitro. Additionally, these transfected cells also develop into the blastocyst stages via SCNT. Based on these in vitro studies, SNCA overexpressed pigs and PRKN KO pigs were successfully produced. Furthermore protein analysis, behavioral changes, and brain imaging analysis were scheduled in both SNCA overexpressed pigs and PRKN KO pigs those were planned to reproduce.ABSTRACT i TABLE OF CONTENTS v LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS x PUBLICATION LISTS xiv PART I. LITERATURE REVIEWS ２ 1. GENOME ENGINEERING ３ 2. SCNT……………………………………………………………….７ 3. TRANSGENIC CLONED PIGS １１ PART II. GENERAL METHODOLOGY １６ CHAPTER I. EXPERIMENTAL PROTOCOLS FOR PORCINE CLONING. １７ 1. CHEMICALS AND MATERIALS １７ 2. OOCYTE PREPARATION １７ 3. DONOR CELL PREPARATION １８ 4. SCNT １９ 5. ET AND PREGNANCY TEST ２７ 6. ANALYSIS OF CLONED ANIMALS ２８ PART III. APPLICATION OF VARIOUS GENE MODULATING TECHNOLOGIES IN IN VITRO ３０ CHAPTER I. PRODUCTION OF CMAH KO PREIMPLANTATION EMBRYOS DERIVED FROM IMMORTALIZED PORCINE CELLS VIA TALEN. ３１ 1. INTRODUCTION ３１ 2. MATERIALS AND METHODS ３３ 3. RESULTS ４１ 4. DISCUSSION ６８ PART IV. PRODUCTION OF GENETIC MODIFIED PIGS ７３ CHAPTER I. TRANSGENIC PIGS OVEREXPRESSING HUMAN SNCA. ７４ 1. INTRODUCTION ７４ 2. MATERIALS AND METHODS ７６ 3. RESULTS ８０ 4. DISCUSSION ９４ CHAPTER II. PRKN KO PIG PRODUCTION VIA TALEN. ９７ 1. INTRODUCTION ９７ 2. MATERIALS AND METHODS ９９ 3. RESULTS １０２ 4. DISCUSSION １０９ PART V. FINAL CONCLUSION １１０ REFERENCES １１３ 국문초록 １３６Docto

돼지에서 Cre-loxP를 이용한 유전자 발현조절 배아생산에 관한 연구

학위논문 (석사)-- 서울대학교 대학원 : 수의학과, 2012. 2. 장구.형질전환 모델 돼지를 만드는 과정에 있어서 조건부 유전자 발현의 필요성이 대두되고 있다. 본 연구에서는 이런 조건부 유전자 발현 시스템인 Cre-loxP 가 돼지의 태아세포에서도 작동을 하는지 여부와 이렇게 만들어진 세포가 핵이 제거된 난자 속에서 reprogramming되어 초기단계의 태아로 발달할 수 있는지 여부를 알아보았다. 본 연구를 위하여 수컷 태아 섬유아세포를 miniature pig로부터 분리하여 배양하였고, 형질전환을 위해 neomycin 저항 유전자가 floxed된 플라스미드인, pCALNL-DsRed를 transfection 하였다. 또한 2주간 750 ㎍/mL의 neomycin을 배양액에 첨가해 주어 형질전환된 세포를 선별하였다. 이렇게 형질전환된 세포는 형광현미경 하에서 아직 빨간형광단백질을 발현하지 않는다. 하지만 한시적으로 존재하는 플라스미드 형태의 Cre 유전자를 transfection해주고 나면 이 섬유아세포는 비로서 빨간형광단백질을 발현하기 시작한다. 이렇게 빨간형광단백질을 발현하는 세포를 somatic cell nuclear transfer (SCNT)의 donor cell로 사용하였다. 총 121개의 난자가 SCNT에 사용되었고, 그 중 76 (62.8%) 개 의 난자에서 난할이 일어났다. 모두 6개의 빨간형광단백질을 발현하는 배반포가 만들어졌다. Floxed되어 있던 neomycin 저항 유전자가 제거가 된 사실은 배반포에서 RT-PCR을 통해서 확인할 수 있었다. 결과적으로 본 연구에서는 Cre-loxP recombination이 miniature돼지의 섬유아세포에서도 정상적으로 작동한다는 사실과 이렇게 형질전환된 세포가 SCNT를 통해서 착상 전 태아발달 과정을 정상적으로 수행한다는 것을 확인하였다. 따라서 Cre-loxP에 의해 conditional 형질전환 복제 돼지의 생산이 가능하다는 사실을 확인하였다.The necessity of conditional gene expression in pigs for making transgenic models is becoming evident. In this study, conditional expression of Cre-loxP in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes to support further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ㎍/mL neomycin for two weeks. The transfected cells did not express red fluorescence under a inverted fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express red fluorescence. The cells expressing red fluorescence were employed for somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%) cleaved. Six blastocysts were developed after SCNT and expressed red fluorescence. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. This study demonstrated that Cre-loxP recombination in miniature pig fibroblasts was successfully conducted and the sequentially transformed cells could develop into the pre-implantation embryo stage via SCNT.Maste

주택 시장권의 공간적 범위에 관한 연구 : 공동주택 분양계약자의 지리적 분포를 중심으로

학위논문(석사) --서울대학교 환경대학원 :환경계획학과(도시 및 지역계획전공),2010.2.Maste

A study between occlusal plane and retromolar pad in Korean

학위논문 (석사)-- 서울대학교 치의학대학원 : 치의학과, 2014. 2. 곽재영.보철 수복시 올바른 교합평면의 회복은 상당히 중요하다. 교합평면 설정 시 참고가 되는 해부학적 구조물들에 대해 많은 연구가 이루어져왔다. 하악의 구후융기는 치조골 흡수가 많이 진행된 경우에도 안정적으로 남아 있어 흔히 사용되는 구조물이다. 교합평면의 높이가 구후융기의 2/3 위치에 존재한다고 알려져 있지만, 이 수치는 서양인들에 관한 연구를 근거로 한다. 본 연구는 한국 유치악자들에서 교합평면의 구후융기에 대한 높이를 측정하고자 시행되었다. 과거 교정치료 경험이 없는 25명의 유치악자들이 선택되었다. 이들의 하악 치열 모형을 제작하였고 모형의 측면을 카메라로 촬영하였다. 컴퓨터 프로그램을 이용해 사진상에서 하악 구치의 협측 교두들과 구후융기의 최고점 및 최저점을 직교좌표계의 좌표로 나타내었다. 협측 교두의 좌표들에 최소자승법을 적용해 교합평면의 후방부를 재현하였다. 재현된 교합평면의 구후융기에 대한 높이를 계산하였다. 결과로서, 구후융기에 대한 상대적인 높이는 평균적으로 0.637(표준편차 0.376)로 나타났다.I. Introduction II. Materials and Methods III. Results IV. Discussion V. Conclusion VI. ReferenceMaste

Virtual sink algorithm for receiver based forwarding in wireless sensor networks

학위논문(석사) --서울대학교 대학원 :전기. 컴퓨터공학부,2007.Maste

스테비올 과생산을 위한 대장균 대사공학

학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2019. 21. Introduction……………………………………………………………………..1 2. Materials and Methods…………………………………………………………6 2.1 Strains, plasmids, and culture conditions…………………………………….6 2.2 Construction of plasmids……………………………………………………..6 2.3 Integration of ent-kaurene pathway into E.coli by CRISPR/Cas9………...…7 2.4 Construction of AtKO N-terminal mutant and SDS-PAGE analysis….……...8 2.5 Genome engineering for improving NADPH availability and measurements of NADP+ and NADPH concentrations…………………………...……………..9 2.6 Construction of artificial self-sufficient trCYP714A2-AtCPR2 fusion protein.9 2.7 Condition of batch fermentation. ……………………………………………10 2.8 Extraction and analysis of products. …………………………………….…10 3. Results………………………………………………………………………….19 3.1 Construction of plasmid-free strain for constitutive production of ent-kaurene……………………………………………………………………..19 3.2 The effect of 5’-UTR on ent-kaurene production…………….…………...…20 3.3 Comparison of kaurene oxidase from S. rebaudiana and A. thaliana for ent-kaurenoic acid production in E.coli……………………………………….…23 3.4 Effect of NADPH/NADP+ ratio on production of ent-kaurenoic acid and stevio………………………………………………………………………...25 3.5 Effects of fusion proteins on steviol production…………………………..…27 3.6 Production of steviol through batch fermentation…………………………...29 4. Discussion………………………………………………………………………31 5. References………………………………………………………………...……33 ABSTRACT IN KOREAN………………………………………………………37MasterAs the incidence of diseases, such as diabetes and obesity, increases, there is an increasing global demand for natural sweeteners to replace sugar. Steviol glycosides, compounds extracted from the herbal plant stevia rebaudiana Bertoni, are valuable in the food and beverage industry because they have intense sweetness and no calories. Recently, engineered Escherichia coli (E.coli) was developed to produce steviol glycosides. However, the conversion rate of ent-kaurenoic acid to steviol, which is aglycone of steviol glycosides, was low due to difficulty of functional expression of cytochrome P450 (P450) in E.coli. In this study, to enhance the biosynthesis of steviol in E. coli, engineering of the P450 proteins was carried out, and genomic editing technology was used, to improve NADPH availability. These enhancements produced approximately 4.3 times more steviol than a control in batch fermentation. In addition, the world’s first ent-kaurene biosynthesis pathway was inserted into the E.coli chromosome to reduce the metabolic burden from plasmids. This constructed strain can produce ent-kaurene without adding antibiotics and inducers. The results of this study can be used to develop biosynthetic strains not only steviol but also other P450-derived metabolites

Knockout mouse

본 발명은 Tph1 또는 Htr2b 넉아웃 마우스 및 이의 제조방법에 관한 것이다. 본 발명의 Tph1 또는 Htr2b 넉아웃 마우스는 타겟 유전자의 넉아웃 효율이 높고, 췌장의 β 세포의 증식능이 감소되어 있으므로 제2형 당뇨 및 대사증후군을 비롯한 다양한 대사 질환 연구에 유용하게 사용될 수 있다