THE STUDY OF ORGANOGENESIS BY MOSAIC PATTERN ANALYSIS

通过马赛克模式分析研究器官发生

• 批准号: 2201500
• 负责人: Philip M Iannaccone
• 金额: $ 17.4万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-12-01 至 1995-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2201500
• 关键词: Mus musculus; cellular oncology; chemical related neoplasm /cancer; disease /disorder model; embryo /fetus; gene expression; genetic markers; genetic strain; genetically modified animals; histochemistry /cytochemistry; histopathology; host neoplasm interaction; immunofluorescence technique; in situ hybridization; isozymes; laboratory rat; liver neoplasms; mathematical model; methylcholanthrene; model design /development; monoclonal antibody; neoplasm /cancer genetics; neoplasm /cancer immunology; neoplastic transformation; nucleic acid probes; nucleic acid repetitive sequence; oncogenes; preneoplastic state; radiotracer; simian virus 40; skin neoplasms; tissue /cell preparation; tissue mosaicism

It is important to the understanding of tumor biology to know if tumors and preneoplastic lesions arise from single cells (i.e., from rare events). Convincing evidence from chimeric mice suggests that chemically induced tumors are clonal in origin. X-linked mosaic mice have supported the growth of subcutaneous fibrosarcomas containing both marker phenotypes present in non- neoplastic tissue, suggesting that these tumors may have a multicellular origin, although there are other explanations for these observations. We propose to attempt a resolution of the question: "Are chemically induced tumors and preneoplastic lesions clonal in origin?" The research involves 4 new systems in which to test the above hypothesis: 1) We have constructed chimeric rats between congenic strains which vary in the expression of class I major histocompatibility antigens. The strain of origin of tissues can be determined in frozen sections using monoclonal antibodies directed to the class I differences in the parental strains. Analysis of tissues from these chimeras has revealed a number of stricking patterns of mosaicism which allow deductions concerning the formation of the organ. We are proposing to examine several models of organogenesis based on the analysis of mosaicism. Liver tumors and preneoplastic, enzyme altered lesions will be induced using the principal hepatocarcinogenic protocols currently available. Tumors and lesions are being analyzed with serial frozen sections, stained with the strain genotype marker of mosaicism, H and E and for enzyme alterations. Reconstruction and quantitative analysis of the sections will be accomplished utilizing a microcomputer based morphometric analysis system that we have developed for this purpose. We will determine whether oncogene activation in chimeric rats occurs in patches or uniformily by in situ hybridization with a probe that recognizes C-Ha-ras. 2) Subcutaneous fibrosarcomas are being induced in Mus musculus (-) Mus caroli interspecific chimeras. The origin of the cells comprising these mosaic animals can be determined in sections utilizing in situ hybridization with a DNA probe which recognizes highly repetitive satellite sequences of Mus musculus. We hope to establish whether host tissue contamination of tumors can account for the presence of both marker phenotypes in electrophoretic analysis of tumors in mosaic animals. 3) Epidermal and subcutaneous tumors are being induced in X-linked mosaic animals (Pgk-1 a/b) where the origin of the cells within the tumor can be determined by electrophoresis of tumor lysates. A combined approach of multiple sampling and tissue culture analysis can provide strong evidence supporting the contention that the neoplastic component of mehtylcholanthrene induced fibrosarcomas are clonally derived. Epidermal tumors are being analyzed by isolating epithelial components from host stromal tissues with a trypsin method we have developed. This analysis will include a critical assessment of the patch size in isolated epidermis of both X-linked mosaic mice and chimeric mice produced from the same strains. 4) We have begun the production of transgenic rodents by the microinjection of oncogenic sequences into pronuclear stage embryos and these procedures will be used to determine whether tumors arise as a result of clonal expansion of given transforming sequences or that oncogenes expression is a necessary but not sufficient condition of tumor formation.

了解肿瘤生物学的理解很重要 肿瘤和肿瘤性病变来自单个细胞（即 罕见事件）。 嵌合小鼠的令人信服的证据表明 该化学诱导的肿瘤起源是克隆的。 X连锁 镶嵌小鼠支持皮下的生长 非纤维肉瘤包含两个标记表型 肿瘤组织，表明这些肿瘤可能具有 多细胞来源，尽管还有其他解释 这些观察。 我们建议尝试解决 问题：“化学诱导的肿瘤和肿瘤肿瘤是否是 病变起源的克隆？”该研究涉及4个新系统 要检验上述假设：1）我们已经构建了 同类菌株之间的嵌合大鼠在 I类主要组织相容性抗原的表达。 应变 可以在冷冻切片中确定组织的起源 针对I类差异的单克隆抗体 父母菌株。 分析这些嵌合体的组织 揭示了许多令人震惊的镶嵌模式，这些模式允许 扣除有关器官形成的扣除。 我们是 提议基于 镶嵌的分析。 肝肿瘤和肿瘤肿瘤， 酶改变的病变将使用原理诱导 目前可用的肝癌方案。 肿瘤和 正在用串行冷冻切片分析病变，染色 用镶嵌性的菌株基因型标记，H和E以及 酶改变。 重建和定量分析 这些部分将通过基于微型计算机来完成 我们为此开发的形态分析系统 目的。 我们将确定是否在 嵌合大鼠发生在斑块中或均匀的原位发生 与识别C-HA-RAS的探针杂交。 2） 在肌肉中诱导皮下纤维肉瘤（ - ） Mus Caroli种间嵌合体。 细胞的起源 包括这些镶嵌动物可以在部分中确定 利用识别的DNA探针的原位杂交 肌肉的高度重复卫星序列。 我们希望 确定肿瘤的宿主组织污染是否可以 说明两个标记表型的存在 镶嵌动物的肿瘤的电泳分析。 3） 表皮和皮下肿瘤正在X连锁中诱导 马赛克动物（PGK-1 A/B），其中细胞的起源 肿瘤可以通过肿瘤裂解物的电泳确定。 多个采样和组织培养的组合方法 分析可以提供支持争论的有力证据 Mehtylcholanthrene诱导的肿瘤成分 纤维肉瘤是克隆派生的。 表皮肿瘤正在 通过从宿主基质中分离上皮成分来分析 我们开发的具有胰蛋白酶方法的组织。 这个分析 将包括隔离的斑块大小的批判性评估 X连锁镶嵌小鼠和嵌合小鼠的表皮 由相同的应变产生。 4）我们已经开始制作 通过微注射的转导啮齿动物的 序列进入前核阶段胚胎和这些过程 将用于确定由于 给定转化序列的克隆扩展或 癌基因表达是必要但不足的条件 肿瘤形成。