SEQUENCE AND POLYMORPHISM OF RHESUS MACAQUE MHC

恒河猴动物模型在HIV疫苗开发和发病机制研究中的效用增加，需要为该物种开发坚实的遗传学和免疫遗传学数据基础设施。相对于人类的数据，有一个缺乏的信息，大规模的基因内容的猕猴MHC和非常少的多态性数据从猕猴I类和II类基因以外。作为一个突出的例子，存在间接证据表明在某些猕猴单倍型上可能存在多达3个I类B基因座和2个I类A基因座，但这尚未通过直接基因组分析证实。因此，在单个实验猕猴中活跃表达的I类基因的数量可能是未知的。一个本质上类似的故事揭示了一些II类基因座，而猕猴MHC中存在的数百个其他基因的结构数据非常少，其中超过三分之一也直接参与免疫反应。这些缺陷提供了实质性的动机，以更好地了解恒河猴的遗传学和免疫遗传学，这可能是有用的设计和艾滋病疫苗的实验中进行类似的免疫缺陷（SIV）和嵌合SIV-HIV（SHIV）的动物模型，使用这种非人灵长类动物物种。为此，我们建议开发一个高质量数据的猕猴主要组织相容性复合体（MHC）的DNA序列。我们进一步建议用广泛的多态性数据（SNP）和更大规模的单倍型变异来注释这个数据串，并且相对于该区域的完整基因内容。一个基本的优先事项将是有效地组织和传播这些数据到感兴趣的科学界。为了开发这个资源，我们建议实现以下具体目标：1）从恒河猴获得一个完整的和高质量的主要组织相容性复合体（MHC）的基因组序列。2)开发和定义一组SNPs和更大规模的单倍型结构，作为与MHC连锁表型相关的作图工具。3)识别和注释猕猴MHC的基因组和多态性，并使这一资源以有用和可口的形式提供给科学界。我们将通过结合三个实验室在猕猴遗传学，基因组学和功能方面的资源和专业知识来实现这些目标。迫切需要分析和监测细胞和亚细胞生物学功能，特别是了解药理学和疾病过程。光学显微镜先前已用于生物样品表征，但具有λ 2的衍射极限分辨率。 然而，扫描近场光学显微镜可以用来实现纳米级分辨率的顺序为1000或更好。该技术结合了原子力显微镜在非接触模式与光学显微镜的属性，通过利用光纤探针尖端，扫描样品表面，同时获取剪切力和光学信号，分别产生地形和光学图像。由二氧化硅制成的光纤微尖已用于可见光波长区域的这项技术，分辨率至少为100 nm。然而，这些光纤不能用于红外线，因为它们不能传输超过2微米的波长。我们建议使用能够进入2-10微米的“指纹”区域的红外传输光纤微尖来开发该技术，在该区域，分子种类可以通过其特征振动吸收带容易地识别。利用最先进的低光学损耗红外光谱技术，开发扫描近场红外显微镜新技术。NRL为各种应用开发的传输光纤。NRL将制造NR-传输微探针，能够探测2-10微米的红外波长区域，这在以前是不可用的。NRL将与范德比尔特的科学家合作，用近场显微镜实现微尖。利用SNIM系统，使用IR光纤微尖，在红外线范围内实现至少100 nm的纳米级光学分辨率。利用扫描近场显微镜（SNIM）在2-10 μ m波长范围内以小于或等于100 nm的分辨率在水环境中研究生物结构（例如活细胞）的光谱成像。将在生物细胞环境中评估探针的光学和形貌分辨率以及光谱成像能力，以分析生物样品中的细胞功能，如膜转运和分子识别过程。这种新的能力对于理解各种药理学和疾病过程至关重要。

The increased utility of the rhesus macaque animal model in both HIV vaccine development and pathogenesis studies necessitates the development of a solid genetics and immunogenetics data infrastructure for this species. Relative to human data, there is a paucity of information about the larger-scale gene content of the macaque MHC and very little polymorphism data from outside of the macaque class I and II genes. As a pointed example, indirect evidence exists that there may be as many as 3 class I B loci and 2 class I A loci on certain macaque haplotypes, but this has not been confirmed through direct genomic analysis. Thus the number of actively expressed class I genes in individual experimental macaque may be unknown. An essentially similar story und4rlies some class II loci, and very little structural data exists about the hundreds of other genes present in the macaque MHC, more than a third of which are also directly involved in the immune response. These deficiencies provide substantial motivation to better understand the genetics and immunogenetics of rhesus macaques that could be useful in the design and conduct of AIDS vaccine experiments in the similar immunodeficiency (SIV) and chimeric SIV-HIV (SHIV) animal models using this non-human primate species. Toward this end, we proposed to develop a DNA sequence of a macaque major histocompatibility complex (MHC) of finished high quality data. We further propose to annotate this data string with extensive polymorphism data (SNP) and larger-scale haplotypic variation, and with respect to the complete gene content of the region. One fundamental priority will be to effective organize and disseminate this data to the interested scientific community To develop this resource, we propose to accomplish the following specific aims: 1) To derive a complete and high quality genome sequence of a major histocompatibility complex (MHC) from the rhesus macaque. 2) To develop and define a set of SNPs and larger scale haplotypes structures useful as mapping tools in the association with MHC-linked phenotypes. 3) To identify and annotate genomes and polymorphisms in the macaque MHC and make this resource available in a useful and palatable form to the scientific community. We will accomplish these aims in part by combining resources and expertise in macaque genetics, genomics, and functions from three accomplished laboratories. There is a critical need for analysis and monitoring of cellular and sub- cellular biological functions, especially for understanding pharmacological and disease processes. Optical microscopy has been previously used for biological sample characterization but has a diffraction limited resolution of lambda 2. However, scanning near-field optical microscopy can be used to achieve nanoscale resolution on the order of lambda30 and better. The technique combines the properties of atomic force microscopy in the non-contact mode with optical microscopy, by utilizing a fiber optic probe tip that scans over a sample surface and simultaneously acquires shear force and optical signals to produce topographical and optical images, respectively. Optical fiber microtips fabricated from silica have been used for this technique in the visible wavelength region with at least 100 nm resolution. However, these fibers cannot be used in the infrared because they do not transmit at wavelengths longer than 2 microm. We propose to develop the technique using infrared-transmitting optical fiber microtips capable of accessing the "fingerprint" region from 2-10 microm, where molecular species can be readily identified by their characteristic vibrational absorption bands/ The goals of this program are: Develop the new technique of Scanning Near Field Infrared Microscopy using state-of-the-art low optical loss IR-transmitting optical fibers which NRL has developed for a variety of applications. NRL will fabricate NR-transmitting microtips capable of probing the 2-10 microm infrared wavelength region that has previously been unavailable. NRL will collaborate with Vanderbilt scientists in the implementation of the microtips with a Near Field Microscope. Demonstrate nanoscale optical resolution at least 100 nm in the infrared with the SNIM system using the IR fiber microtips. Utilizing the scanning near-field microscopy (SNIM) in the 2-10 microm wavelength region to investigate spectroscopic imaging of biological structures such as living cells at less than or equal too 100 nm resolution in an aqueous environment. The optical and topographical resolution as well as spectroscopic imaging capability of the probes will be evaluated in a biological cellular environment, to analyze cellular functions such as membrane transport and molecular recognition processes in biological samples. This new capability will be critical for the understanding of a wide variety of pharmacological and disease processes.