Production of cloned embryos using PRNP-knockout fibroblasts

학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2017. 2. 장구.Prion is one of the membrane protein of most kinds of cells, but its conformational changes and accumulations induce transmissible spongiform encephalopathies (TSE), known as mad cow disease, in mammalian species. The first production of prion-free cattle was reported in 2007 by homologous recombination, but a wide range of physiological and pathological functions of prion is still unknown, and the gene targeting using homologous recombination is time and cost consuming process. Recently, the powerful nucleases, transcription activator-like effector nuclease (TALEN) and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9, have been widely used for site-specific DNA recombination. They have a wide range of applicability from plants to mammalians, and the efficiency was known to be high. In this study, the prion protein (PRNP)-disrupted bovine cell lines have been established which are readily applicable to generate PRNP-free embryos, and functional studies on prion were conducted. For the first step of gene targeting in bovine cells, immortalization of bovine fibroblasts was tried to enhance efficiency of in vitro study. Two genes, B lymphoma moloney murine leukemia virus (Mo-MLV) insertion region 1 homolog (BMI1) and human telomerase reverse transcriptase (hTERT), were introduced into bovine fibroblasts by random integration, and proliferation of the BMI1+hTERT introduced cells was maintained after 60 passage in vitro, although the naïve fibroblasts and the BMI1-introduced cells were arrested after 20 and 40 passages, respectively. In the reconstructed embryos via somatic cell nuclear transfer (SCNT), the immortalized cells were successfully reprogramed, and the embryos were developed up to preimplantation stages. The expression level of p53, one of tumor suppressor, was maintained at low in the whole period of in vitro culture, and the telomerase activity was significantly increased at passage 60 compared with passage 20. These expression alterations compared with other cell lines are considered as supporting factors for cell proliferation in the immortalized cells. Nextly, customized TALEN was designed to eliminate PRNP expression. The TALEN pair targeting exon 3 of PRNP gene and the reporter-expressing vectors were transferred into the immortalized cells, first. By co-transfection with the reporter vector, it was capable of enrichment of mutation–positive cells. After sorting the transfected cells, they were seeded to generate monoclonal cell population, and each cell populations were collected and analyzed. Among the isolated cell lines, about 67 % of cell lines were T7E1 positive and about 19 % were biallelic mutations. In the sequence analysis, various types of mutations including deletions and insertions were observed, and the mutation patterns were consistently maintained in the cloned embryos. Although the mRNA expression of PRNP was not observed in the PRNP-knockout embryos, they were fully developed until blastocysts stage with similar developmental rate with controls. Expression analysis of the PRNP-related genes was opposed to the previous study, and further investigation should be needed to reveal the gene expressions according to the cell line and mutation type. The normal type of prion expression was also evaluated in different bovine organs and ovarian follicular structures according to estrus cycles. As expected, the prion expression was strong in spinal cord compared with the expressions in others (heart, liver, lung, spleen, kidney, and ovary). In the ovarian follicles, the prion expression altered depending on follicular stages. All kinds of corpus luteum expressed prion strongly, but the expression was weakly detected in the dominant follicles larger than 8 mm of diameter. In addition to the PRN-knockout experiments, the PRNP-targeting knockin vector containing homology arms of the TALEN domains was successfully integrated into bovine genomic DNA. The integration of exogenous sequence was confirmed through PCR analysis, and the mRNA expression of PRNP was eliminated by the result of the knockin mutation targeting PRNP sequence. Following successful knockout and knockin via TALEN in bovine cells, genomic mutation using CRISPR/Cas9 system was induced in the bovine cells. As a screening research of CRISPR/Cas9 system on bovine cells, knockout of exogenous DNA was induced. To evaluate the knockout efficiency of CRISPR/Cas9, the enhanced green fluorescent protein (eGFP)-expressing cell line was established: the piggyBac-mediated eGFP integration was induced in bovine fibroblasts, and its cloned embryo was transferred to collect transgenic fetus. The green fluorescence expression was confirmed under fluorescence microscope. The CRISPR/Cas9 expressing vectors targeting exogenous eGFP sequence were transferred into the eGFP fetal fibroblasts by single electroporation stimuli, and about 40 % of eGFP cells have lost their fluorescence signals. The disruption of exogenous gene did not affect to the capability of knockout cells to generate blastocysts in vitro. Finally, the CRISPR/Cas9 targeting bovine PRNP gene was designed and transfected with the reporter vector. The PRNP mutation rates induced by CRISPR/Cas9 were 66 % and 70 % among the monoclonal colonies of the immortalized cells. Moreover, more than a half of the monoclonal cell lines were mutation-positive also in the normal bovine fibroblasts. The mutation patterns were confirmed through sequence analysis and fluorescent PCR (fPCR), and they were consistent in the cloned embryos generated by SCNT. After transfer the PRNP-mutant cloned embryo into the recipient, successful implantation was confirmed at day 40 through ultrasound imaging. In conclusion, the bovine immortalized cells with exogenous BMI1 and hTERT were stably established and applied for bovine in vitro studies. In addition to that, mutations on PRNP were effectively induced by introduction of TALEN and CRISPR/Cas9, respectively, and PRN-knockout embryos were developed normally in vitro despite a lack of understanding of prion. These genetic tools are expected to be applicable to generate transgenic animals which have a relatively large diversity of targeted mutations.PART I. LITERATURE REVIEW 19 1. IN VITRO IMMORTALIZATION OF BOVINE CELLS 20 2. GENE TARGETING IN CATTLE 28 3. PRION DISEASES 35 PART II. GENERAL METHODOLOGY 36 1. REAGENTS 37 2. OOCYTE COLLECTION AND IN VITRO MATURATION 37 3. SOMATIC CELL NUCLEAR TRANSFER (SCNT) 40 4. IN VITRO CULTURE (IVC) OF EMBRYOS 43 5. IN VITRO CULTURE OF FIBROBLASTS 45 6. REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION 48 PART III. IMMORTALIZATION OF BOVINE CELLS 49 CHAPTER I. IMMORTALIZATION OF BOVINE CELLS 50 1. INTRODUCTION 50 2. MATERIALS AND METHODS 53 3. RESULTS 59 4. DISCUSSION 72 PART IV. KNOCKOUT VIA TALEN IN BOVINE CELLS 75 CHAPTER I. PRNP-KNOCKOUT AND KNOCKIN VIA TALEN IN IMMORTALIZED CELLS 76 1. INTRODUCTION 76 2. MATERIALS AND METHODS 78 3. RESULTS 81 4. DISCUSSION 89 CHAPTER II. EXPRESSION OF PRNP AND RELATED GENES 91 1. INTRODUCTION 91 2. MATERIALS AND METHODS 93 3. RESULTS 95 4. DISCUSSION 102 PART V. KNOCKOUT VIA CRISPR/CAS9 IN BOVINE CELLS 105 CHAPTER I. MUTATION SCREENING OF CRISPR/CAS9 IN BOVINE CELLS 106 1. INTRODUCTION 106 2. MATERIALS AND METHODS 108 3. RESULTS 115 4. DISCUSSION 121 CHAPTER II. PRNP-KNOCKOUT VIA CRISPR/CAS9 IN BOVINE CELLS 123 1. INTRODUCTION 123 2. MATERIALS AND METHODS 124 3. RESULTS 126 4. DISCUSSION 134 FINAL CONCLUSION 136 REFERENCES 139 국문초록 161Docto

The Influence of Workplace Learning on Employee Competence Improvement in Small and Medium-Sized Enterprises

본 연구는 중소기업에서 근로자들의 일터학습 참여가 직무능력향상에 어떠한 영향을 미치고, 또 개인특성 및 학습지원환경이 일터학습에 어떠한 영향을 미치는지를 실증적으로 분석하였다. 본 연구는 전국의 중소기업을 업종, 규모, 지역에 따라 표집하였으며, 최종적으로 중소기업 근로자 497명의 응답을 분석에 활용하였다. 연구 결과, 개인특성과 학습지원환경은 근로자들의 일터학습 참여에 정적으로 유의한 영향을 미치고, 일터학습 참여는 중소기업 근로자들의 직무능력향상에 정적으로 유의한 영향을 미치는 것으로 나타났다. 특히 중소기업의 학습지원환경이 근로자 일터학습 참여에 미치는 영향력은 개인특성이 일터학습 참여에 미치는 영향력보다 큰 것으로 드러났다. 또한 중소기업 근로자들의 직무능력은 구조화되고 계획화된 교육훈련 참여와 동료 및 선후배 등과의 상호작용을 통한 자기주도적 학습활동에 의해 향상될 수 있는 것으로 밝혀졌다. 이러한 연구결과를 바탕으로 본 연구에서는 이론적·실무적 시사점과 향후 연구를 위한 방향을 제시하였다.This study pursues to analyze the influence of workplace learning on employee competence improvement, and investigate the influences of personal characteristics and supportive learning environment on workplace learning. This study employed a set of sample collected from 497 employees through a proportional stratified sampling. The results showed that personal characteristics and supportive learning environment have significantly positive impact on workplace learning, and workplace learning has significant and positive impact on employee competence improvement. It is found that employee competence can be improved by workplace learning such as planned and structured programs as well as social interactions and self-initiated learning activities

Performance comparison and evaluation of interferon-gamma assay kit for bovine tuberculosis diagnosis

In Korea, bovine tuberculosis (bTB) is a representative zoonotic disease that causes considerable economic loss. In determining the positive bTB, the ELISA method for examining the amount of interferon- gamma (IFN-γ) is included in Koreas diagnostic standard method. Recently, commercially available BIONOTE TB-Feron ELISA Plus (TB-Feron Plus) that detects IFN-γ has been introduced. However, since the scientific basis for the performance is limited, we evaluated performance by comparing it with the results of another IFN-γ ELISA assay kit (BOVIGAM Ⓡ ) certified by Office International des Epizooties. In our research, 42 positive blood samples preliminarily tested with a tuberculin skin test and/or BOVIGAM Ⓡ and 54 negative blood samples collected from three bTB free farms were subjected to IFN-γ assay using the TB-Feron Plus and the BOVIGAM Ⓡ , respectively. The result shows that the sensitivity, specificity and accuracy were 81.0% (34/42), 100% (54/54), 91.7% (88/96) in TB-Feron Plus kit and 78.6% (33/42), 100% (54/54), 90.6% (87/96) in BOVIGAM Ⓡ kit, respectively. Moreover, the overall accordance percentage of the two kits was 99.0% (95/96) and there was almost perfect agreement between two assays (Kappa=0.977, P＜0.0001). Furthermore, additional studies confirmed that elevated lymphocyte numbers in blood did not interfere with the results of the TB-Feron Plus kit. And, delayed time from sampling to culture decreased the optical density (OD) value. Therefore, we concluded that the TBFeron Plus kit was not inferior to BOVIGAM Ⓡ in performance. High lymphocyte numbers in blood did not impact on TB-Feron Plus results, while delayed time before culture interfered with OD value.N