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Title: | 單體紅色螢光蛋白質基因轉殖小鼠及豬之產製及其生物醫學應用之潛能分析 Generation of Transgenic Mice and Pigs Harboring the DsRed-Monomer Reporter Gene and Characterization of Potentials for Biomedical Applications |
Authors: | Chih-Jen Chou 周志任 |
Advisor: | 吳信志 |
Keyword: | 單體紅色螢光蛋白質,紅色螢光蛋白質基因轉殖小鼠,紅色螢光蛋白質基, DsRed-monomer,red fluorescence protein,transgenic mice,transgenic pigs, |
Publication Year : | 2014 |
Degree: | 博士 |
Abstract: | 螢光蛋白質可提供追踪標定的功能,在生物體中追踪基因表達及細胞行為相關的研究。螢光蛋白質是一個非常珍貴的工具,它應用在生命科學領域的追踪標定功能有許多年了。單體紅色螢光蛋白質(DsRed-Monomer)是屬於一個長波長的紅螢光蛋白質(RFP),鑑於其具備幾個優點:在37℃時快速折疊完成形成功能性蛋白質、更明亮之特性、低能量之螢光激發光譜及在動物組織中觀察時產生較少之自發性螢
光,所以單體紅色蛋白質遂於近數年間,迅速發展成為熱門之追踪用標定蛋白質。因此,本研究的目的旨在探討產製單體紅色螢光蛋白質基因轉殖小鼠與基因轉殖豬及其生物醫學應用之可行性評估。 本研究目標是產製單體紅色螢光蛋白質基因轉殖小鼠及基因轉殖豬,將含有巨細胞病毒CMV 增強子及雞的β-肌動蛋白啟動子驅動的質體DNA,透過原核基因顯微注射技術之策略,將之引入小鼠胚/或豬胚。攜帶單體紅色螢光蛋白質基因之基因轉殖小鼠和基因轉殖豬已被成功產製,其外源單體紅色螢光蛋白質基因且被證實確可透過性腺被傳承至下一個世代;性腺傳承率分別約為52.72%及43.59%(58/110 和17/39)。進一步將基因轉殖小鼠和基因轉殖豬犧牲及並針對包括腦、心臟、肺、肝、胰腺、胃、脾臟、小腸、腎臟、睾丸、子宮、卵巢、肌肉及眼等組織或器官分別進行採樣後,再透過激發光激發(激發波長520-540 nm,發射波長580-650 nm)及濾片過濾後,且分別被確認均有表現紅螢光蛋白質之事實。此外,進一步藉由組織學檢測和蛋白質印跡分析等技術,針對各個組織進行驗證結果,且證實確有表現紅螢光蛋白質之情事。藉由組織病理學分析技術,針對前述轉基因小鼠組織器官(包括:腦、心臟、肺、肝、胰腺等等)切片進行驗證之結果,顯示彼等源自基因轉殖小鼠胰臟組織之腺泡細胞,確實呈現彼等胞質內的嗜酸性線狀物質附加之情事。此外,進一步驗證包括藉由血液學,血清學及血漿生化值等驗證結果分別均可提供做為評估身體狀況和動物健康之一個重要參數。基因轉殖小鼠與野生型小鼠在經過血液檢查比較後,在淋巴細胞、單核細胞、丙胺酸胺基轉移酶(ALT)和穀草轉胺酶(AST)等,雖略有影響;惟藉由驗證結果證明,在轉基因豬與野生型豬二者間的組織學、血液檢查及電腦斷層掃瞄並無顯著差異情事。進一步試驗針對帶有單體紅螢光蛋白基因之轉基因母豬,在懷孕之第70 天收集其並完成分離其羊水中之前驅幹細胞,再經流式細胞儀(FACS)完成分選出彼等細胞所表現之表面抗原,分析結果證明,羊水前驅幹細胞表現CD44 及CD90,惟並無CD4a 及CD31表現之情事。此外,該細胞在體外證明可以分化成為脂肪細胞、軟骨細胞和成骨細胞。 綜上合述,本研究試驗成功產出單體紅色螢光蛋白質之轉基因小鼠與轉基因豬,並確認所有取樣器官具有紅色螢光蛋白質表現及將其基因性腺傳承予後代。此等表現有之紅色螢光蛋白質轉基因小鼠和轉基因豬可以做為宿主並應用於其它顏色螢光標記的細胞,俾供後續研究探討細胞與細胞之間涉及微環境間相互作用機制,且能夠提供最佳的紅色螢光蛋白質標記的細胞和組織在發育生物學、再生醫學及異種 移植之研究。 Fluorescent proteins provide much more powerful targets to track gene expression and cellular behaviors in the study of model organisms. These proteins have served as invaluable tools fitting to those purposes related to genetic engineering studies for many of the coming years. The monomeric red fluorescent protein (RFP) is a long wavelength of fluorescent proteins,it expresses specific molecular characteristics, such as: rapid maturation at 37°C, brighter and lower energy excitation spectrum, and less automonous fluorescence of animal tissues etc. Base on the fact that this molecule behavior to be rapid maturation, the RFP has since been used particulars for tracking target in the recent years. The purpose of this study has been focused to is generate transgenic mice and pigs harboring the DsRed-Monomer reporter gene and to evaluate the potentials for biomedical applications. To meet purposes described above, the initial studied were made to generate the DsRed-monomer transgenic mouse and pig by means of pronuclear microinjection of the transgene driven by the cytomegalovirus CMV enhancer/chicken beta-actin promoter. Transgenic mice and pigs harboring a gene of monomeric red fluorescent protein were generated and further confirmed to be transmitted their modified genome into their offspring. Both of the founder mice and/or pigs were further comfirmed to be able to transmit their modified genotype into their first generation, with their germ-line transmission rates arroud 52.72% (58/110) and 43.59% (17/39) in these transgenic lined mice and pigs respectively. These transgenic mice and/or pigs appeared to be ubiquitously expressed their red fluorescent protein found in those sampling organs and/or tissues (including: brain、 eye、 tongue、 heart、 lung、 liver、 spleen、 pancreas、 stomach、 small intestine、 large intestine、 kidney、 testis、 uterus、 ovary、 muscle and hoofs etc.) were all microscopically confirmed by excitation light wavelength and filter (excitation 520-540 nm, emission 580-650 nm). Furthermore, such expression was reconfirmed by histological assay and western blot analyses of each various tissues. In the respect of histopathology assay, the mouse tissue sections dissected from organs or tissue (including: brain、 eye、 tongue、 heart、 lung、 liver、 spleen、 pancreas、 stomach、 small intestine、 large intestine、 kidney、 testis、 uterus、 ovary、 muscle and eye etc.) of wild-type and/or RFP transgenic mice, the results revealed the following distinct histological discrepancy in pancreases of transgenic mice with additional intra-cytoplasmic eosinophilic threadlike materials in acinar cells compared with those sample for the wild-type mice. Both of those values related to hematology, serology and/or plasma biochemistry value appeared to be important parameter(s) for further assess emendation of physical conditions and animal health. The transgenic mice were minor affected in these parameters relate to lymphocytes、 monocytes、 alanine aminotransferase (ALT) and aspartate aminotransferase (AST), when comparisons were made these found in the wild-type mouse. However, there was no significant differences found between these of control pigs and Ds-red bearing transgenic pigs when histology、blood examinations and also the computed tomography were conducted. Furthermore, when progenitor stem cells derived from those of amniotic fluid of Ds-red bearing fetuses at day 70 of pregnant sows. These cells cells confirmed being able to be could differentiated into adipocyte、osteoblast even the chondrocyte in vitro. In addition, results generated affect the FACS (fluorescence-activated cell sorting) assessment indicate that these cells have been equipped with strong potential to express CD44, CD90 while not to express either CD4a and/or CD31 gene(s). Taken together, DsRed-Monomer transgenic mice and pigs were successfully produced, and confirmed the RFP expression on all of the sampling organs and transmitted the phenotype to their offspring. These red fluorescent mice and pigs can serve as a host for other fluorescent-labeled cells in order to study cell-microenvironment interactions, and can provide optimal red-fluorescent-labeled cells and tissues for research in developmental biology, regenerative medicine, and xenotransplantation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18368 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生物科技研究所 |
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