吾等研究目標著重於應用體細胞核移殖（somatic cell nuclear transfer, SCNT, 或稱 cloning, 複製）結合gene-targeted技術產製基因剔除家兔，供研究心血管疾病機制、藥物篩選及治療之改進。目前為止，仍無任何具多能性（pluripotent）之兔胚幹細胞或基因標的轉殖兔被成功報導。透過SCNT產製與基因轉殖供核細胞完全一致之基因轉殖動物，提供現今發展基因標的動物最可信並最具潛能之技術。吾等近期之研究已自卵丘細胞成功產製複製兔，來自密西根大學及Evergen Biotechnologies, Inc.之共同合作研究團隊，亦成功建立進行SCNT所需之家兔CRP (C-reactive protein, 與心血管疾病極重要關連之基因) 標的基因供核纖維母細胞系。家兔係被認為所有複製動物中最具挑戰性物種之一，複製效率至今仍難以突破，主要原因推測受卵母細胞(recipient oocyte)之品質、複製胚細胞數過少及再程序化過程發生缺陷等因素所導致。故本計畫擬透過： 1) 排卵時間及位置對卵母細胞品質之影響；2) 添加Trichostatin A改變複製胚核再程序化過程中之乙醯化程度，提高複製效率；3) 與四倍體胚、孤雌致活胚或受精胚聚合後，提高重組複製胚細胞數；4) 進而探討以此改良系統產製CRP基因剔除家兔之可行性。
國內在複製兔相關研究方面，尚未有任何體細胞複製家兔成功之實例，本計畫主持人在此等動物體細胞複製研究領域已有數年嫻熟之經驗, 將結合與美國頂尖研究團隊卓越之gene-targeting及 SCNT核心技術，共同建立一基因剔除家兔之創新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, 並將領導心血管疾病於藥物篩選及治療改進之臨床研究邁入另一新的里程碑。
Abstract: Cardiovascular disease (CVD) is the leading cause of death and a major cause of disability worldwide. The study of the cardiovascular system has benefited significantly from the use of transgenic mouse models; however, these 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.
The long-term goal of our study is using somatic cell nuclear transfer (SCNT) and the gene-targeting technologies to produce gene-targeted transgenic rabbits as CVD and other human disease models for the study of the disease mechanisms and development of therapies. 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 transgenic animals. Previously we successfully cloned rabbits from cumulus cells, and more recently in embryonic blastomere. We have also successfully targeted the C-reactive protein (CRP) gene, a promising candidate gene to predictive of human cardiovascular disease and heart attacks, in rabbit fibroblast cells. However, rabbit is considered as one of the most challenge species to be cloned and the efficiency is extremely low. Major reasons may be attributed to the quality of recipient oocyte, low cell number of cloned embryo and incomplete epigenetic reprogramming etc.
In this research project, we propose to increase the efficiency of nuclear transfer by determining: a) the age effect of rabbit oocyte for somatic cell nuclear reprogramming, b) the effect of trichostatin A (TSA), an inhibitor of histone deacetylase, to enhance cloning efficiency, c) alternative approaches to improve cloned embryo quality by aggregate multiple NT or tetraploid and parthenogenetic embryos, and d) finally the feasibility to generate knockout rabbit with CRP targeted cell lines. Through this proposed project, a feasible method to produce gene targeted transgenic rabbits will be developed, upon the success this technology will not only provide a novel animal model system for developing drugs specific for human CVD, but also establish a new approach to generate various gene targeted transgenic rabbit lines. Some part of proposed research is being performed in the Institute of Biotechnology at NTU.
We are fully confident that our research would reach a world level in the area of human CVD research and novel drug discovery with support of this NTU Frontier and Innovative research fund.