Kao Betty R, McColl Bradley, Vadolas Jim
Cell and Gene Therapy Research Group, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Road, Parkville, 3052, Melbourne, VIC, Australia,
Methods Mol Biol. 2015;1227:323-43. doi: 10.1007/978-1-4939-1652-8_18.
Bacterial artificial chromosome (BAC) reporter cell lines are generated through stable transfection of a BAC reporter construct wherein the gene of interest is tagged with a reporter gene such as eGFP. The large capacity of BACs (up to 350 kb of genomic sequence) enables the inclusion of all regulatory elements that ensure appropriate regulation of the gene of interest. Furthermore, the reporter gene allows the expression of the gene of interest to be readily detected by flow cytometry. Cell lines can also be easily cultured for extended periods with minimal cost. These features of BAC reporter cell lines make them highly amenable for use in high-throughput screening of large drug libraries for compounds that induce the expression of the gene of interest. This chapter describes a method for generation of BAC reporter cell lines that are suitable as cellular assay systems in high-throughput screening. Briefly, this method involves (A) generation of cell clones stably transfected with a BAC reporter construct, (B) selection of "candidate" cell clones based on the responsiveness to known inducers, (C) confirmation of the integrity of the BAC reporter construct integrated within the candidate clones, and (D) assessment of the developmental regulation of the BAC reporter construct. As an example, we describe the generation of a BAC reporter cell line containing the human β-globin locus modified to express γ-globin as eGFP for use as a cellular reporter assay for screening of drugs that can reactivate expression of developmentally silenced γ-globin for the treatment of β-hemoglobin disorders.
细菌人工染色体（BAC）报告细胞系是通过稳定转染BAC报告构建体产生的，其中感兴趣的基因用报告基因如增强绿色荧光蛋白（eGFP）进行标记。BAC的大容量（高达350 kb的基因组序列）使得能够包含所有确保对感兴趣基因进行适当调控的调控元件。此外，报告基因使感兴趣基因的表达能够通过流式细胞术轻松检测到。细胞系也可以以最小的成本轻松长期培养。BAC报告细胞系的这些特性使其非常适合用于高通量筛选大型药物文库，以寻找能够诱导感兴趣基因表达的化合物。本章描述了一种生成BAC报告细胞系的方法，该细胞系适合作为高通量筛选中的细胞检测系统。简而言之，该方法包括（A）用BAC报告构建体稳定转染产生细胞克隆，（B）基于对已知诱导剂的反应性选择“候选”细胞克隆，（C）确认整合在候选克隆中的BAC报告构建体的完整性，以及（D）评估BAC报告构建体的发育调控。作为一个例子，我们描述了一种BAC报告细胞系的生成，该细胞系包含经过修饰以表达γ-珠蛋白作为eGFP的人β-珠蛋白基因座，用作细胞报告检测，用于筛选能够重新激活发育沉默的γ-珠蛋白表达以治疗β-血红蛋白疾病的药物。
