Novel method to produce transgenic rabbits

描述（由申请人提供）：拟议研究的长期目标是生产转基因/敲除（KO）兔作为人类心血管疾病（CVD）和其他疾病的模型动物。兔一直被认为是比小鼠和大鼠更好的CVD模型动物，但传统的DNA显微注射方法效率低，且无法获得胚胎干细胞系，阻碍了转基因/KO兔的生产。我们提出了一种解决上述问题的新方法：利用基因操纵体细胞进行核转移（NT）来产生转基因/KO克隆。在I期研究中，我们选择c反应蛋白（CRP）作为靶基因。我们建议制造CRP基因敲除的克隆兔。具体目的包括：(1)鉴定2-3个特性良好的兔成纤维细胞系；(2)在不同的传代上对这些细胞系进行基因操作，产生CRP敲除细胞系；(3)利用KO细胞系和非KO细胞系制备克隆兔胚胎；(4)转移克隆胚胎制备CRP-KO-NT家兔。这些努力将为商业化生产转基因/KO兔作为CVD和其他人类疾病研究的模型动物奠定基础。目前在美国还没有这样的转基因兔子设施，我们相信我们提出的项目将首次使兔子，这种有价值但研究不足的模型动物，可供全国和全世界的研究人员使用。

DESCRIPTION (provided by applicant): The long term objectives of proposed study is to produce transgenic/knockout(KO) rabbits as model animals for human cardiovascular disease (CVD) and other diseases. Rabbit has been long recognized a better CVD model animal than mouse and rat, however transgenic/KO rabbit production is hampered largely by low efficiency rate of the conventional DNA microinjection method and the unavailability of embryonic stem cell lines. We propose a novel solution to aforementioned problems: use genetically manipulated somatic cells for nuclear transfer (NT) to produce transgenic/KO clones. In Phase I study, we choose C-reactive protein (CRP) as the targeted gene. We propose to produce CRP knockout cloned rabbits. The specific aims include: (1) identify 2-3 well-characterized rabbit fibroblast cell lines; (2) genetically manipulate these cell lines at different passages, generating CRP knockout cell lines; (3) produce cloned rabbit embryos using KO cell lines vs. non-KO cell lines; and (4) transfer cloned embryos to produce CRP-KO-NT rabbits. These efforts will establish the foundation for commercially producing transgenic/KO rabbits as model animals for CVD and other human disease studies. Such transgenic rabbit facility is not available in the United States at this moment and we believe our proposed project will for the first time make rabbit, the valuable however under-studied model animal, available to researchers throughout the country and worldwide.