USE OF BAC TRANSGENIC ANIMALS FOR ANALYSIS OF GENE EXPRESS & FUNCTION IN THE CN

使用 BAC 转基因动物进行基因表达分析

• 批准号: 8169112
• 负责人: NATHANIEL HEINTZ
• 金额: $ 1.16万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169112
• 关键词: Affinity Chromatography; Animals; Antibodies; Atlases; Bacterial Artificial Chromosomes; Biochemical; Brain; Cell Nucleus; Cells; Cellular Morphology; Complex; Computer Retrieval of Information on Scientific Projects Database; Control Animal; DNA; Data; Development; Epitopes; Escherichia coli; Funding; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Genes; Genetic Transcription; Genets; Genome; Genomics; Grant; Growth Factor; Imagery; Institution; Invertebrates; Laboratories; Location; Macromolecular Complexes; Mammalian Cell; Mediating; Methods; Modification; Mus; Mutation; Nature; Neuraxis; Nucleotides; Pattern; Peptides; Preparation; Procedures; Proteins; Research; Research Personnel; Resolution; Resources; Site; Source; Stimulus; Synapses; System; Techniques; Time; Transgenic Animals; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; base; cell type; chelation; cytokine; gene function; granule cell; homologous recombination; in vivo; interest; method development; novel; protein complex; protein distribution; release factor; response; transcription factor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The development of methods for the simple and accurate manipulation of Bacterial Artificial Chromosomes (BACs) in my laboratory has allowed the utilization of an alternative and highly efficient strategy for analysis of CNS specific genes (Heintz 2000). This approach is based on two simple facts: large genomic DNA fragments (100KB) are in most instances expressed independent of the site of integration into the genome of transgenic mice; inclusion of epitope tags and marker proteins into endogenous loci of invertebrate genes has in most cases not altered the patterns of expression of these genes or the localization of their encoded products within the cell. To take advantage of this information, a homologous recombination system was established in E. coli that allows for preparation of BACs with highly precise modifications. Using this system, it is possible to create mutations in BACs that range from single nucleotide changes to deletions of tens of kilobases to insertions of marker genes of several kilobases. One can, therefore, construct BACs that allow very rapid analysis of the expression pattern of the gene of interest, the localization of its encoded product, high-resolution visualization of the morphology of cells expressing the gene, and determination of the projection patterns of these cells. Mice made using these techniques also carry epitope tagged proteins that can be used for affinity purification of complexes carrying the protein of interest. The use of epitope tags for determination of the subcellular distribution of proteins in invertebrates and in cultured mammalian cells is very well established. Because of the precision of homologous recombination in E. coli, it is quite simple to introduce an epitope tag into the protein encoded by the gene of interest in the BAC at the same time that one introduces the marker genes. Since a variety of epitope tags and their cognate antibodies are now available commercially, one has a wide range of options from which to choose. Although the introduction of an epitope tag into the protein can in some cases change its subcellular distribution, this is relatively infrequent and usually can be overcome by changing the location of the tag within the protein. Since preparation of useful antibodies for a protein of interest is often an expensive and long-term project, the ability to detect the epitope tagged protein in vivo offers a very efficient and useful alternative. In trying to interpret CNS expressed gene function, localization of its encoded product, or correlation of its subcellular distribution in different cell types or under different conditions can provide crucial information. Obviously, the spectrum of functions one might consider is significantly different for proteins located in the nucleus than those present at the synapse! Furthermore, the redistribution of the protein in response to a stimulus can also be quite informative. For example, there are many well characterized transcriptional responses that involve regulated release of factors from cytoplasmic complexes and their entry into the nucleus in response to growth factors, cytokines, etc. (unpublished data). The ability to obtain this type of information in an efficient manner using epitope tags presents a significant advantage over the time consuming preparation of sufficiently useful antibodies to the native protein for these studies. The development of peptide tags for affinity purification is also of great utility. We have, for example, inserted the 6XHis tag into the Zipro1 locus in BAC transgenic animals for isolation of Zipro1 containing transcription complexes from cerebellar granule cells. It is now possible to utilize Ni+ chelation affinity chromatography to characterize the Zipro1 complexes using whole brain extracts from the BAC transgenic mice as has been very successfully done for His-tagged transcription factors in cultured mammalian cells. This strategy can be extended for purification of any macromolecular complex from any cell type in the brain using the BAC transgenic approach. Since the results from the animal carrying the epitope tagged protein can be directly compared to control animals, background from the purification procedure can be identified readily. While affinity purification methods are not yet fully developed for this purpose, the use of BAC transgenic animals for this purpose is a major advance over current method for identifying protein complexes that exist in vivo. When combined with the advanced mass spectrometric methods carried out in the Chait Laboratory for protein identification, this approach offers a novel and highly efficient alternative to traditional biochemical techniques. Heintz, N. (2000). "Analysis of mammalian central nervous system gene expression and function using bacterial artificial chromosome-mediated transgenesis." Hum Mol Genet 9(6): 937-43. Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N."A gene expression atlas of the central nervous system based on bacterial artificial chromosomes" Nature 425(2003)917-25

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 在我的实验室中简单，准确地操纵细菌人造染色体（BAC）的方法的开发允许利用一种替代性且高效的策略来分析CNS特定基因（Heintz 2000）。 这种方法基于两个简单的事实：在大多数情况下，大型基因组DNA片段（100KB）独立于整合到转基因小鼠的基因组中；在大多数情况下，将表位标签和标记蛋白纳入无脊椎动物基因的内源性基因座没有改变这些基因的表达模式，也没有改变其编码产物在细胞中的定位。 为了利用这些信息，在大肠杆菌中建立了同源重组系统，该系统允许制备具有高度精确修改的BAC。 使用该系统，可以在BAC中产生突变，范围从单核苷酸变化到数十千酶的缺失到插入多个千倍酶的插入。 因此，可以构建BAC，从而可以非常快速地分析感兴趣基因的表达模式，其编码产物的定位，表达基因的细胞形态的高分辨率可视化以及确定这些细胞的投影模式。使用这些技术制成的小鼠还携带表位标记的蛋白质，可用于携带感兴趣蛋白质的复合物的亲和力纯化。 使用表位标签来确定无脊椎动物和培养的哺乳动物细胞中蛋白质的亚细胞分布。 由于大肠杆菌中同源重组的精确性，因此将一个表位标签引入了由BAC中感兴趣的基因编码的蛋白质中，同时引入了标记基因。由于现在商业上可以使用各种表位标签及其同源抗体，因此可以选择各种选择。尽管在某些情况下，将表位标签引入蛋白质可以改变其亚细胞分布，但这相对较少，通常可以通过更改蛋白质中标签的位置来克服。由于为感兴趣的蛋白质制备有用的抗体通常是一个昂贵且长期的项目，因此在体内检测表位标记的蛋白质的能力提供了非常有效且有用的替代方案。在试图解释中枢神经系统表达的基因功能，其编码产物的定位或在不同细胞类型中或在不同条件下的亚细胞分布的相关性可以提供至关重要的信息。显然，位于核中的蛋白质的功能范围与突触中存在的蛋白质明显不同！此外，响应刺激的蛋白质的重新分布也可能非常有用。例如，有许多特征性的转录反应涉及细胞质复合物中因子的调节释放，并响应生长因子，细胞因子等，将其进入细胞核（未发表的数据）。使用表位标签以有效的方式获取这种信息的能力在这些研究中对天然蛋白的足够有用的抗体的准备就具有显着优势。用于亲和力纯化的肽标签的开发也很有用。例如，我们将6xHis标签插入了BAC转基因动物中的Zipro1基因座中，以分离含有小脑颗粒细胞的转录复合物的Zipro1。现在，可以利用Ni+螯合亲和力色谱法使用BAC转基因小鼠的全脑提取物来表征Zipro1复合物，就像在培养的哺乳动物细胞中对His标记的转录因子的成功所做的那样。可以使用BAC转基因方法扩展该策略以纯化大脑中任何细胞类型的任何大分子复合物。由于携带表位标记蛋白的动物的结果可以直接与对照动物进行比较，因此可以轻松地识别出纯化程序的背景。尽管为此目的尚未完全开发亲和力纯化方法，但在此目的中使用BAC转基因动物是鉴定体内存在的蛋白质复合物的主要进步。当与蛋白质鉴定的Chait实验室中采用的高级质谱方法结合使用时，该方法提供了一种新型且高效的替代传统生化技术的替代方法。 Heintz，N。（2000）。 “使用细菌性人造染色体介导的转基因的哺乳动物中枢神经系统基因表达和功能分析。” Hum Mol Genet 9（6）：937-43。 Gong S，Zheng C，Doughty ML，Losos K，Didkovsky N，Schambra UB，Nowak NJ，Nowak NJ，Joyner A，Leblanc G，Hatten Me，Heintz N。