STABLE EXPRESSION OF TRANSFERRED NUCLEIC ACIDS IN MAMMALIAN CELLS

基于遗传学的疗法的一个最终目标涉及转移 核酸对骨髓再生细胞的稳定作用 编码基因在患者生命周期中的表达。这 这种方法最终可能对治疗流行的遗传性疾病有用 包括地中海贫血和镰状细胞贫血在内的疾病。然而， 在基因治疗完全实现之前，涉及 核酸在哺乳动物体内的转移及其调控表达 必须定义单元格。转导核糖核酸的稳定表达 哺乳动物细胞中的酸仍然是一个鲜为人知的现象。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。绿色荧光蛋白已成功地作为一种 瞬时基因表达的报告分子，但报告稳定 GFP的表达是稀疏的。因此，我们工作的目标是 了解生产细胞系所需的条件 持久的、高水平的GFP表达。中国仓鼠卵巢(CHO)细胞 用编码绿色荧光蛋白和新霉素的质粒DNA 磷酸转移酶(NEO)盒。流式细胞术和目测法 监测在有无细菌存在情况下培养的细胞 G418介导的选择压力在池中稳定表达 在非选择性培养液中培养12周以上。这一观察结果 表明GFP本身并不会给经济增长带来不利影响 哺乳动物细胞。我们的目标是培养出造血细胞系和 在缺乏GFP的情况下稳定表达GFP的原代造血细胞 选择性压力。一旦确定，这些细胞将允许进一步 基因稳定表达的研究。

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.