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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。在我的实验室中，发展了简单而准确地操作细菌人工染色体(BAC)的方法，使我们能够利用一种替代的、高效的策略来分析中枢神经系统特有的基因(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(100kb)的表达与整合到转基因小鼠基因组中的位置无关；在大多数情况下，将表位标签和标记蛋白包括到无脊椎动物基因的内源位置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。为了利用这一信息，在大肠杆菌中建立了一个同源重组系统，允许制备具有高精度修饰的BAC。使用这个系统，可以在BAC中产生突变，范围从单核苷酸变化到数万个碱基的缺失，再到几千个碱基的标记基因的插入。因此，人们可以构建能够非常快速地分析目的基因的表达模式、其编码产物的定位、高分辨率可视化表达基因的细胞的形态以及确定这些细胞的投射模式的BAC。使用这些技术制造的小鼠也携带表位标记蛋白，可用于亲和纯化携带感兴趣蛋白的复合体。利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。由于大肠杆菌中同源重组的精确性，在引入标记基因的同时，将表位标签引入到BAC中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以在商业上获得，人们有各种各样的选择可供选择。虽然将表位标签引入蛋白质在某些情况下可以改变其亚细胞分布，但这种情况相对较少，通常可以通过改变标签在蛋白质中的位置来克服。由于为感兴趣的蛋白质制备有用的抗体通常是一个昂贵和长期的项目，因此在体内检测标记的表位蛋白质的能力提供了一个非常有效和有用的替代方案。在试图解释CNS表达的基因功能时，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，人们可能考虑的位于核内的蛋白质的功能光谱与存在于突触中的蛋白质显著不同！此外，蛋白质对刺激的反应重新分配也可以提供相当多的信息。例如，有许多具有良好特征的转录反应，涉及对生长因子、细胞因子等的反应，从细胞质复合体中调节因子的释放并进入细胞核(未发表的数据)。与耗时制备足够有用的天然蛋白抗体相比，使用表位标签以有效的方式获得这类信息的能力具有显著的优势。用于亲和纯化的多肽标签的开发也是非常有用的。例如，我们已经将6XHis标签插入到BAC转基因动物的Zipro1基因座中，以从小脑颗粒细胞中分离出含有Zipro1转录复合体的基因。现在可以利用镍离子螯合亲和层析从BAC转基因小鼠的全脑提取液中鉴定Zipro1复合体，就像在培养的哺乳动物细胞中非常成功地对His标记的转录因子所做的那样。这一策略可以扩展到使用BAC转基因方法从大脑中任何类型的细胞中提纯任何大分子复合体。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，因此可以很容易地识别纯化过程中的背景。虽然亲和纯化方法还没有完全发展到这一目的，但使用BAC转基因动物来鉴定存在于体内的蛋白质复合体的方法是一个重大进展。当与柴特实验室进行蛋白质鉴定的先进的质谱学方法相结合时，该方法为传统的生化技术提供了一种新颖而高效的替代方法。Heintz，N.(2000)。使用细菌人工染色体介导的转基因技术分析哺乳动物中枢神经系统的基因表达和功能。哼哼摩尔基因9(6)：937-43。基于细菌人工染色体的中枢神经系统基因表达图谱《自然》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