STABLE EXPRESSION OF TRANSFERRED NUCLEIC ACIDS IN MAMMALIAN CELLS

哺乳动物细胞中转移核酸的稳定表达

• 批准号: 6161921
• 负责人: J L MILLER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6161921
• 关键词: CHO cells; DNA; flow cytometry; gene expression; green fluorescent proteins; hematopoietic stem cells; phosphotransferases; transfection; transfection /expression vector

One eventual goal of genetics-based therapies involves the transfer of nucleic acids to bone marrow repopulating cells and the stable expression of the encoded gene over the lifetime of the patient. This approach may eventually be useful for the treatment of prevalent genetic diseases including the thalassemias and sickle-cell anemia. However, before gene therapy can be fully realized, basic paradigms involving the transfer of nucleic acids and their regulated expression in mammalian cells must be defined. The stable expression of transferred nucleic acids in mammalian cells remains a poorly understood phenomenon. We have begun the study of stable green fluorescent protein (GFP) gene expression in mammalian cells. GFP has been used successfully as a reporter molecule for transient gene expression, but reports of stable GFP expression are sparse. Thus, the objective of our work was to understand the conditions required to produce cell lines that exhibit durable, high-level GFP expression. Chinese hamster ovary (CHO) cells were transfected with plasmid DNA encoding both GFP and neomycin phosphotransferase (neo) cassettes. Flow cytometric and visual monitoring of the cells cultured in the presence and the absence of G418-mediated selective pressure revealed stable expression in pools after more than 12 weeks in nonselective medium. This observation suggests that GFP itself does not bestow a growth disadvantage in mammalian cells. Our goal is to produce hematopoietic cell lines and primary hematopoietic cells that stably express GFP in the absence of selective pressure. Once identified, these cells will permit further study of stable gene expression.

基于遗传学的治疗的一个最终目标涉及转移 核酸对骨髓再生细胞的作用， 在患者的一生中编码的基因的表达。 这 这种方法最终可能有助于治疗流行的遗传性 地中海贫血和镰状细胞性贫血等疾病。然而，在这方面， 在基因治疗完全实现之前， 核酸的转移及其在哺乳动物中的调节表达 必须定义单元格。 转移核酸的稳定表达 哺乳动物细胞中的酸仍然是一种知之甚少的现象。 我们 已经开始研究稳定的绿色荧光蛋白（GFP）基因 在哺乳动物细胞中表达。 GFP已成功用作 瞬时基因表达的报告分子，但报告稳定的 GFP表达稀少。 因此，我们工作的目标是 了解产生细胞系所需的条件， 持久的高水平GFP表达。 中国仓鼠卵巢（CHO）细胞 用编码GFP和新霉素的质粒DNA转染 磷酸转移酶（neo）盒。流式细胞术和目视检查 监测在存在和不存在以下物质的情况下培养的细胞： G418介导的选择压力显示池中的稳定表达 在非选择性培养基中培养超过12周后。 该观察结果 表明GFP本身并不赋予生长劣势， 哺乳动物细胞 我们的目标是生产造血细胞系， 原代造血细胞，其在缺乏GFP的情况下稳定表达GFP， 选择性压力 一旦确定，这些细胞将允许进一步的 稳定基因表达的研究。