摘要:體細胞核移置(Somatic cell nuclear transfer, SCNT)或稱動物複製(Animal cloning),具有將已分化之細胞再程序化(reprogramming)至相當於受精胚階段全能性(totipotent)能力之細胞。以SCNT配合基因標的(gene targeting)技術產製人類疾病模式動物,提供關鍵平台以進行各種疾病機制研究、藥物篩選與治療之改進。心血管疾病為現今世界各國之主要死因或導致行為能力障礙之最大因素。根據衛生署98年最新資料統計,心血管疾病(心臟疾病占10.6%、腦血管疾病占7.3%)在台灣為僅次於惡性腫瘤死亡率之主要死因,故此領域為醫學研究之極重要範疇。一般相關研究大多以基因標的小鼠為重要基礎,然就人類心血管生理而言,小鼠卻非一適當之動物模式。另一方面,家兔之心血管解剖構造、生理及脂質代謝等特性,與人類具有極高之相似性,故家兔即為研究人類心血管疾病不惶多讓之最佳動物模式。
透過SCNT產製與基因標的供核細胞完全一致之動物,提供現今發展基因標的動物最可信並最具潛力之技術,而再程序化不完全所造成的複製率低下亦為此等技術目前面臨的挑戰之一。近期吾等團隊之研究已於台灣大學自卵丘細胞(cumulus)成功產製體細胞複製家兔,來自University of Michigan 及 Renova Life Inc. 之共同合作研究團隊亦將CRP基因(C-reactive protein, 與心血管疾病極重要關連之基因)透過基因標的方式成功地轉殖入家兔纖維母細胞中,與心血管疾病重要之WHLL 突變家兔細胞亦應用作為實驗對照組。因此,本3年期計畫將首先著重於研究複製家兔之核再程序化過程及其機制之了解,從而改善整體複製效率,進一步地,冀望以CRP 基因轉殖細胞產製CRP基因標的家兔,以供人類心血管疾病相關研究及治療之最佳動物模式。家兔被認為是所有複製動物中最具挑戰物種之一,目前為止,其低複製效率仍難以突破,主要原因推測係因卵母細胞遺傳來源及品質、複製胚之後生遺傳再程序化不完全(incomplete reprogramming)或發生缺陷所致,故本計畫擬 1). 首先建立家兔胚胎發育過程中重要基因- Oct4 及組蛋白乙醯化 (histone acetylation)之後生遺傳調控模式做為改善複製胚胎核再程序化之依據;2). 並透過不同方式包括:a) 家兔供卵細胞之遺傳背景,b) 添加 histone deacetylase (HDAC) inhibitors (如: Trichostatin A 及 Scriptaid) , 及 c) 原核互換(pronuclear exchange)方式,改變複製胚胎後生遺傳模式,促進核之再程序化,並進而提升複製效率;進一步地,3). 探討以此改良系統產製WHLL 或 CRP-基因標的動物之可行性。
本計畫主持人在動物體細胞複製研究領域已有多年豐富經驗, 近期業已於台灣大學成功建立研究平台,產製出台灣第一隻體細胞複製家兔,且將結合先前與美國頂尖研究團隊卓越之基因標的核心技術,延續國際合作並共同建立一創新之人類心血管疾病研究模式。透過本計畫之執行,除有助於國內在體細胞複製及心血管疾病研究邁向卓越外, 並將推展心血管疾病之臨床研究於藥物篩選及改進治療技術邁入另一嶄新之里程碑。
Abstract: Somatic cell nuclear transfer (SCNT, also called cloning) is the technology capable of reprograms a
differentiated somatic cell to regain totipotent capacity. Combine SCNT with gene-targeted and cell
therapy represents a great promise to study many human diseases. Cardiovascular disease (CVD) is the
leading cause of death and a major cause of disability worldwide. Although the study of CVD has greatly benefited from the use of gene-targeted transgenic mouse models, however, the small rodents do not accurately reflect human cardiovascular physiology. Rabbit proved to be an ideal animal model for CVD research, considering its many similarities to humans in cardiovascular anatomy, physiology, and lipid metabolism, as well as elasticity and ease of handling for many types of studies.
To date, due to the lack of germline transmitting rabbit embryonic stem cells, no gene targeted transgenic rabbits have been produced. Animal cloning via SCNT using the transgenic cells would result in all identical transgenic offspring, provide a great potential to generate the gene-targeted animals. Recently we successfully cloned rabbits from cumulus cells at National Taiwan University. Our collaborators at the University of Michigan and Renova Life Inc. also successfully targeted the C-reactive protein (CRP) gene, the great model to predictive of human cardiovascular disease and heart attacks, in rabbit fibroblast cells. Moreover, the natural mutant WHLL rabbit fibroblast cell lines for CVD research are created for SCNT control as well. Therefore, the final goal of this proposal will focus on using CRP-gene target cells to produce the CRP-gene targeted rabbits through SCNT as CVD models to study the mechanisms of human disease and development of therapies. However, rabbit consider as one of the most challenge species to be cloned, the cloning efficiency is remain extremely low. Major reasons may be attributed to the
genotype of animal, incomplete epigenetic reprogramming and low cell number in cloned embryo before
implantation.
Therefore, in this 3-year of research project, we first propose to 1) establish the important epigenetic profile of Oct4 and histone acetylation during rabbit embryogenesis, which provides the fundamental base for diagnosis the successful of nuclear reprogramming in cloned rabbit embryos. 2) Improve cloning efficiency by following strategies: a) using hybrid rabbit as source of recipient oocytes, b) modify epigenetic reprogramming of cloned embryos with HDAC inhibitors (i.e. Trichostatin A and Scriptaid), c) alternatively approaches by pronuclear exchange. Epigenetic reprogramming events in cloned rabbit embryos will be performed to understand the mechanisms of nuclear reprogramming via SCNT in rabbit. Finally, 3) we will test the feasibility of generate cloned rabbits from WHLL or CRP gene-targeted fibroblast cells. We are confident that through this proposed project, a feasible method to produce gene targeted transgenic rabbits will be developed and it will not only establish a new platform to generate genetargeted transgenic animals, but also provide a novel system for study the mechanisms and developing drugs for human CVD.