Engineered Cell Lines for Regulatory Protein Analysis

用于调节蛋白分析的工程细胞系

• 批准号: 6742250
• 负责人: JOHN A STAMATOYANNOPOULOS
• 金额: $ 10万
• 依托单位: REGULOME CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742250
• 关键词: alkaline phosphatase; biotechnology; cell line; chromatin; crosslink; deoxyribonuclease I; erythroleukemia; gene expression; genetic manipulation; genetic regulation; genetic regulatory element; globin; nucleic acid chemical synthesis; nucleoproteins; protein quantitation /detection; protein sequence; technology /technique development; transcription factor; transfection /expression vector

DESCRIPTION (provided by applicant): The overall aim of this Phase I research proposal is to develop a novel cell-based system to enable efficient recovery and quantitative analysis of the regulatory proteins complexed at a specific, well-characterized human cis-regulatory site. The proposal comprises 3 Specific Aims: Specialized vectors will be synthesized (Specific Aim 1). Each vector comprises 3 main components: (i) a well-characterized cis-regulatory element (human beta-globin 5'HS2); (ii) a quantitative selectable marker (secreted alkaline phosphatase); and (iii) paired chromatin insulator elements (either human HS5 or chicken HS4). A qualitative selectable marker (neo) is also included. Two vectors will be produced that are identical save for the choice of insulator sequence. These vectors will be stably transfected into human erythroleukemia cells (K562), a well established system, and high copy cell lines will be selected (Specific Aim 2). The high-copy cell lines will be further examined to confirm functional activity of the HS2 element through quantitative assessment of DNaseI hypersensitivity (McArthur et al. 2001). Preliminary studies will be undertaken in a selected multi-copy line to determine the optimal conditions for excision of the intact HS2 nucleoprotein complex from crosslinked chromatin (Specific Aim 3). The vectors are specifically designed to take advantage of the fact that DNA sequences immediately flanking the protein-binding core regions of DNaseI hypersensitive sites are accessible to the action of sequence-specific restriction endonucleases in the context of nuclear chromatin. In the vector, the HS2 element is flanked by restriction sites for rare cutters that will excise a specific approximate 600bp fragment. Purification of this fragment and its associated proteins may be thus effected solely on the basis of size and solubility fractionation. Fractionation over sucrose gradients has the added advantage of providing a clean reagent for downstream mass spectrometric studies. Such studies are envisioned to form the basis of a follow-on Phase II proposal.

描述（由申请人提供）：本I期研究提案的总体目标是开发一种新型的基于细胞的系统，以有效回收和定量分析在特定的、充分表征的人顺式调节位点复合的调节蛋白。该提案包括3个具体目标：将合成专门的载体（具体目标1）。每个载体包含3个主要组分：（i）充分表征的顺式调节元件（人β-珠蛋白5 'HS 2）;（ii）定量选择标记（分泌的碱性磷酸酶）;和（iii）成对的染色质绝缘子元件（人HS 5或鸡HS 4）。还包括定性选择标记（neo）。将产生两个矢量，除了绝缘子序列的选择之外，它们是相同的。将这些载体稳定转染至人红白血病细胞（K562）（一种完善的系统）中，并选择高拷贝细胞系（特定目标2）。将进一步检查高拷贝细胞系，以通过定量评估DNaseI超敏反应来确认HS 2元件的功能活性（麦克阿瑟等人，2001）。将在选定的多拷贝系中进行初步研究，以确定从交联染色质中切除完整HS 2核蛋白复合物的最佳条件（特异性目标3）。载体被特别设计以利用这样的事实，即紧邻DNaseI超敏感位点的蛋白质结合核心区侧翼的DNA序列在核染色质的背景下可接近序列特异性限制性内切酶的作用。在该载体中，HS 2元件的侧翼是用于切割特异性约600 bp片段的罕见切割酶的限制性位点。因此，该片段及其相关蛋白的纯化可以仅基于大小和溶解度分级来实现。在蔗糖梯度上的分级具有额外的优点，即为下游质谱研究提供清洁的试剂。预计这些研究将成为第二阶段后续提案的基础。