| 条件性缺氧诱导因子-1α RNAi转基因小鼠模型的建立 |
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| 引用本文： | 戚华兵,宋瑞华,杜晓兰,赵 玲,苏 楠,刘道诚,李福兵,陈 林.条件性缺氧诱导因子-1α RNAi转基因小鼠模型的建立[J].生物工程学报,2008,24(1):27-32. |
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| 作者姓名： | 戚华兵 宋瑞华 杜晓兰 赵 玲 苏 楠 刘道诚 李福兵 陈 林 |
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| 作者单位： | 第三军医大学第三附属医院野战外科研究所全军创伤中心,创伤、烧伤与复合伤国家重点实验室,重庆,400042 |
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| 基金项目： | 国家重点基础研究发展项目(973计划)(No. G1999054203 and No. 2005CB522604); 国家自然科学基金资助项目(No. 30370318)。 |
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| 摘 要： | 缺氧诱导因子1a (hypoxia inducible factor-1 a, HIF-1 a)是细胞在缺氧等条件下稳定表达的具有转录活性的蛋白,通过与多种靶基因调控区的缺氧反应元件(hypoxia response element, HRE)结合, 调控靶基因表达, 使机体对缺氧、缺血等病理生理过程产生适应性反应。为从整体动物水平研究HIF-1 a的作用, 需要建立HIF-1 a相关遗传修饰小鼠。分别针对HIF-1 a mRNA序列的两个靶位点合成两对互补的寡核苷酸链, 构建可诱导的RNA干扰真核表达载体HIF-AB和HIF-CD。分别将CRE重组酶真核表达载体CRE-ERT2与HIF-AB或HIF-CD转染入RAW264.7细胞, 筛选得到稳定表达CRE-ERT2与HIF-AB, 或CRE-ERT2与HIF-CD的稳定细胞系。在用4-HT诱导去除上述细胞系中HIF-AB或HIF-CD所含的Neo基因后, 用CoCl2诱导HIF-1 a表达, 采用半定量RT-PCR检测HIF-AB或HIF-CD对HIF-1 a 基因表达的影响。结果发现干扰载体(HIF-AB和HIF-CD)对HIF-1 a mRNA序列的沉默效果分别为85%和72%。选择干扰效率较高的表达载体HIF-AB经显微注射获得HIF-1 a基因敲低小鼠模型, 经PCR以及测序验证获得2个转基因阳性小鼠(Founders, G0代)。G0代雄鼠与FVB/N雌鼠交配后获得2只F1代(first filial generation)转基因阳性小鼠, 经与EIIA-Cre转基因小鼠交配, 得到EIIA-Cre; HIFRNAiflox/+小鼠, RT-PCR结果显示, EIIA-Cre; HIFRNAiflox/+小鼠肝、肺、肾等组织的HIF-1 a mRNA水平明显降低, 分别约为正常对照的44%、38.2%和23.5%。该小鼠模型的建立为进一步研究HIF-1 a的功能及作用机制提供了新的手段。
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| 关 键 词： | 缺氧诱导因子-1a 发夹状小干涉RNA 转基因小鼠 |
| 收稿时间： | 2006-12-19 |
| 修稿时间： | 2007-01-17 |
Generation of Inducible HIF-1a RNAi Transgenic Mice |
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| Huabing Qi,Ruihua Song,Xiaolan Du,Ling Zhao,Nan Su,Daocheng Liu,Fubing Li and Lin Chen.Generation of Inducible HIF-1a RNAi Transgenic Mice[J].Chinese Journal of Biotechnology,2008,24(1):27-32. |
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| Authors: | Huabing Qi Ruihua Song Xiaolan Du Ling Zhao Nan Su Daocheng Liu Fubing Li and Lin Chen |
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| Institution: | Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China;Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University; State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing 400042, China |
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| Abstract: | |
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| Keywords: | HIF-1 a shRNA transgenic mice |
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