Production of New Models for Biomedical Research by RNAi

通过 RNAi 生产生物医学研究新模型

• 批准号: 7037693
• 负责人: MARK E WESTHUSIN
• 金额: $ 21.83万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-22 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7037693
• 关键词: Lentivirus; RNA interference; biotechnology; disease /disorder model; gene induction /repression; genetic manipulation; genetically modified animals; goats; method development; model design /development; myostatin; recombinant virus; transfection /expression vector

DESCRIPTION (provided by applicant): The long term goal of the proposed research is to produce transgenic animals which can be used as models for biomedical research by employing RNA interference to knock-down expression of specific genes. The ability to genetically engineer animals has proven to be an invaluable tool for biomedical research. Transgenic mice have been extremely useful for investigating human health issues including cancer, diabetes, cardiovascular disease, AIDS, kidney disease, and immunology, just to name a few. Other animal models are also important for research involving human health and welfare, as mice are not completely representative of human genetics and physiology. Valuable research leading to new information useful for developing treatments for (or the prevention of) human diseases can be derived from comparative studies involving the use of different animal species for biomedical research. Transgenic animals representing species other than mice would be extremely useful; unfortunately, the ability to produce transgenic animals other than mice has proven difficult and in many cases cost-prohibitive. In addition, the utilization of embryonic stem cells (ES cells) for gene targeting by homologous recombination to create models for human development and disease has thus far proven unsuccessful in species other than mice. In recent times, several exciting breakthroughs promise efficient methods to allow the production of transgenic animals representing a wide variety of different species. The utilization of lentiviral vectors to produce transgenic mice has proven to be extremely effective. We have recently demonstrated that lentiviral vectors can also be efficiently used to produce transgenic bovine embryos. In addition, techniques involving RNA interference (RNAi) have now been developed which allow silencing of specific genes by introducing short hairpin RNAs (shRNAs) into the genome, thus bypassing the need for homologous recombination. Finally, the utilization of recombinant lentiviral vectors in combination with techniques involving RNAi has now been successfully utilized to create transgenic mice in which specific genes were successfully targeted for silencing. These techniques should be applicable to a wide variety of mammalian species. The specific aim of this proposal is to develop and utilize a lentiviral vector system to produce transgenic goats encoding shRNAs targeting myostatin (GDF 8). Single-cell embryos will be produced, and then infected with a recombinant lentiviral vector carrying gene constructs coding for the shRNAs targeting myostatin. Alternatively, a recombinant lentiviral vector will be used to deliver shRNAs targeting myostatin into caprine fibroblasts followed by nuclear transfer to produce cloned transgenic embryos in which myostatin has been targeted for silencing. Embryos will be transferred into synchronized recipient females for the production of transgenic offspring. The resulting offspring will be analyzed to determine if they are transgenic and exhibit the appropriate phenotype. We expect to produce transgenic goats in which expression of myostatin has been effectively silenced by RNAi. We anticipate these animals will exhibit increased muscle growth and mass when compared to controls. Success of this project will lead to techniques which can be applied to a wide variety of different animal species, greatly expanding their usefulness as models for research involving human health and disease.

描述（由申请人提供）：拟议研究的长期目标是产生转基因动物，可以通过采用RNA干扰来敲破特定基因的表达来将其用作生物医学研究的模型。事实证明，具有基因研究动物的能力是生物医学研究的宝贵工具。转基因小鼠对于研究人类健康问题非常有用，包括癌症，糖尿病，心血管疾病，艾滋病，肾脏疾病和免疫学，仅举几例。其他动物模型对于涉及人类健康和福利的研究也很重要，因为小鼠并不完全代表人类遗传学和生理学。有价值的研究导致新信息可用于开发（或预防）人类疾病的治疗方法，可以从涉及将不同动物物种用于生物医学研究的比较研究中得出。代表小鼠以外的物种的转基因动物将非常有用。不幸的是，事实证明，生产小鼠以外的其他转基因动物的能力很困难，并且在许多情况下，成本良好。此外，迄今为止，在小鼠以外的其他物种中，胚胎干细胞（ES细胞）通过同源重组为人类发育和疾病创建模型的基因靶向基因。最近，一些令人兴奋的突破有效的方法有效的方法允许产生代表各种不同物种的转基因动物。事实证明，使用慢病毒载体产生转基因小鼠的利用非常有效。我们最近证明，慢病毒载体也可以有效地用于产生转基因牛胚胎。此外，现在已经开发了涉及RNA干扰（RNAI）的技术，从而通过将短发夹RNA（SHRNA）引入基因组中，从而使特定基因沉默，从而绕开了对同源重组的需求。最后，现在已成功地利用了重组慢病毒载体与涉及RNAi的技术结合使用，以创建转基因小鼠，其中成功靶向用于沉默的特定基因。这些技术应适用于多种哺乳动物。该提案的具体目的是开发和利用慢病毒矢量系统来生产编码靶向肌生抑制素的shRNA的转基因山羊（GDF 8）。将产生单细胞胚胎，然后用重组慢病毒载体载有基因构建体，用于靶向肌生抑素的shRNA。或者，将使用重组慢病毒载体将靶向肌抑制素的shRNA递送到囊蛋白成纤维细胞中，然后进行核转移，以产生克隆的转基因胚胎，在该克隆的转基因胚胎中，将肌化素靶向用于沉默。胚胎将被转移到同步的受体女性中，以产生转基因后代。将分析由此产生的后代，以确定它们是否是转基因并表现出适当的表型。我们期望产生转基因山羊，其中肌抑制素的表达有效地被RNAi沉默。我们预计，与对照组相比，这些动物将表现出增加的肌肉生长和肿块。该项目的成功将导致技术可以应用于各种不同的动物物种，从而大大扩展了它们作为涉及人类健康和疾病的研究模型的实用性。