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应用日益增加，因此有必要为该物种建立坚实的遗传学和免疫遗传学数据基础设施。相对于人类数据，关于猕猴MHC的更大规模基因含量的信息缺乏，来自猕猴I类和II类基因以外的多态性数据很少。作为一个尖锐的例子，有间接证据表明，在某些猕猴单倍型上可能存在多达3个I类B位点和2个I类a位点，但这并没有通过直接的基因组分析得到证实。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。在一些II类基因座的基础上，也存在着一个本质上类似的故事，关于猕猴MHC中存在的数百个其他基因的结构数据很少，其中超过三分之一的基因也直接参与免疫反应。这些缺陷为更好地了解恒河猴的遗传学和免疫遗传学提供了重要的动力，这可能有助于在使用这种非人类灵长类动物的类似免疫缺陷（SIV）和嵌合SIV- hiv （SHIV）动物模型中设计和开展艾滋病疫苗实验。为此，我们建议建立猕猴主要组织相容性复合体（MHC）的DNA序列，以获得高质量的完整数据。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该数据串，并对该区域的完整基因含量进行注释。为了开发这一资源，我们建议实现以下具体目标：1)获得恒河猴主要组织相容性复合体（MHC）的完整和高质量的基因组序列。2)开发和定义一组snp和更大规模的单倍型结构，作为与mhc相关表型相关的制图工具。3)鉴定和注释猕猴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。我们将在一定程度上通过结合三个有成就的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。对细胞和亚细胞生物学功能的分析和监测，特别是对药理学和疾病过程的理解，是一个至关重要的需要。光学显微镜以前曾用于生物样品表征，但其衍射极限分辨率为λ 2。然而，扫描近场光学显微镜可以实现在lambda30数量级甚至更高的纳米级分辨率。该技术结合了非接触模式下原子力显微镜与光学显微镜的特性，利用光纤探针尖端扫描样品表面，同时获取剪切力和光信号，分别产生地形和光学图像。由二氧化硅制成的光纤微针尖已用于该技术，其分辨率至少为100nm。然而，这些光纤不能用于红外线，因为它们不能在波长超过2微米的地方传输。我们建议使用红外传输光纤微针尖开发技术，该技术能够进入2-10微米的“指纹”区域，在该区域，分子物种可以通过其特征振动吸收带很容易地识别。该计划的目标是：利用NRL为各种应用开发的最先进的低光损耗红外传输光纤，开发扫描近场红外显微镜的新技术。NRL将制造能够探测2-10微米红外波长区域的nr发射微尖，这在以前是不可用的。NRL将与范德比尔特大学的科学家合作，用近场显微镜实现微针尖。使用红外光纤微针尖演示SNIM系统在红外中至少100 nm的纳米级光学分辨率。利用扫描近场显微镜（SNIM）在2-10微米波长区域研究生物结构的光谱成像，如在水环境中小于或等于100纳米分辨率的活细胞。探针的光学和地形分辨率以及光谱成像能力将在生物细胞环境中进行评估，以分析生物样品中的膜运输和分子识别过程等细胞功能。这种新的能力对于理解各种各样的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.