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

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 在我的实验室中，开发了简单而准确地操作细菌人工染色体（BAC）的方法，这使得可以利用替代的高效策略来分析CNS特异性基因（Heintz 2000）。 这种方法基于两个简单的事实：在大多数情况下，大的基因组DNA片段（100 KB）的表达与转基因小鼠基因组的整合位点无关;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位点并没有改变这些基因的表达模式或其编码产物在细胞内的定位。 为了利用这些信息，我们在大肠杆菌中建立了同源重组系统。大肠杆菌，允许制备具有高度精确修饰的BAC。 使用该系统，有可能在BAC中产生突变，其范围从单个核苷酸变化到数十个内切酶的缺失到几个内切酶的标记基因的插入。 因此，可以构建BAC，其允许非常快速地分析感兴趣的基因的表达模式、其编码产物的定位、表达基因的细胞的形态的高分辨率可视化以及这些细胞的投影模式的确定。使用这些技术制备的小鼠还携带表位标记的蛋白质，其可用于亲和纯化携带感兴趣蛋白质的复合物。 使用表位标签来确定蛋白质在无脊椎动物和培养的哺乳动物细胞中的亚细胞分布是非常成熟的。 由于E.在大肠杆菌中，在引入标记基因的同时，将表位标签引入BAC中由目的基因编码的蛋白质中是相当简单的。由于各种表位标签和它们的同源抗体现在可商购获得，因此人们具有广泛的选择范围。虽然在某些情况下，将表位标签引入蛋白质中可以改变其亚细胞分布，但这相对不常见，通常可以通过改变标签在蛋白质中的位置来克服。由于制备用于感兴趣的蛋白质的有用抗体通常是昂贵且长期的项目，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，或其在不同细胞类型或不同条件下的亚细胞分布的相关性可以提供关键信息。显然，位于细胞核中的蛋白质的功能谱与位于突触中的蛋白质的功能谱是显著不同的！此外，蛋白质响应于刺激的重新分布也可以提供相当多的信息。例如，有许多充分表征的转录应答，其涉及调节因子从细胞质复合物的释放以及它们响应于生长因子、细胞因子等进入细胞核（未发表的数据）。使用表位标签以有效方式获得这种类型的信息的能力相对于耗时制备足够有用的天然蛋白质抗体以用于这些研究具有显著的优势。用于亲和纯化的肽标签的开发也具有很大的实用性。例如，我们已经将6XHis标签插入BAC转基因动物的Zipro 1基因座中，用于从小脑颗粒细胞中分离含有Zipro 1的转录复合物。现在可以利用Ni+螯合亲和色谱法，使用BAC转基因小鼠的全脑提取物表征Zipro 1复合物，这在培养的哺乳动物细胞中已非常成功地用于His标记的转录因子。该策略可以扩展用于使用BAC转基因方法从脑中的任何细胞类型纯化任何大分子复合物。由于携带表位标记的蛋白质的动物的结果可以直接与对照动物进行比较，因此可以容易地鉴定纯化过程的背景。虽然亲和纯化方法尚未完全开发用于此目的，BAC转基因动物用于此目的是一个主要的进步，目前的方法，用于鉴定体内存在的蛋白质复合物。当与Chait实验室用于蛋白质鉴定的先进质谱方法相结合时，这种方法为传统的生物化学技术提供了一种新颖且高效的替代方法。 Heintz，N.（2000年）的第10/2000号决议。“使用细菌人工染色体介导的转基因分析哺乳动物中枢神经系统基因表达和功能。《分子遗传学》9（6）：937-43。 Gong S，Zheng C，Doughty ML，Losos K，Didkovsky N，Schambra UB，Nowak NJ，Joyner A，Leblanc G，哈滕ME，Heintz N.“基于细菌人工染色体的中枢神经系统基因表达图谱”Nature 425（2003）917-25

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