FUNCTION OF FIBROBLAST GROWTH FACTOR GENE IN MAMMALIAN EMBRYOGENESIS

越来越多的证据表明，碱性成纤维细胞生长因子9b- 以及相关蛋白在胚胎诱导过程中起着重要作用。 在哺乳动物中，有四个基因(a-fgf、k-fgf、int-2和fgf-5)被 发现了与b-成纤维细胞生长因子有广泛序列相似性的，以及更多是 很可能存在。我们有兴趣研究这些基因在 哺乳动物的早期发育。 我们的第一组具体目标是确定每个 这些基因通过原位rna在发育中的小鼠胚胎中表达。 杂交和蛋白质定位，并从这些信息到 开始建立一个关于它们在发育中的作用的假说 可能是吧。然而，由于细胞的阶段和组织特异性功能 这些不同的生长因子将取决于阶段-和组织- 对于它们各自受体的特异性，我们还建议研究 成纤维细胞生长因子受体(S)在胚胎发育中的表达为了 要实现这些目标，我们必须为其中的每一个都提供可用的探测器 老鼠的基因。到目前为止，成纤维细胞生长因子家族中唯一具有 已在INT-2中克隆。我们采用了各种技术， 利用聚合酶链式反应(聚合酶链式反应)克隆所有 小鼠成纤维细胞生长因子家族的已知成员，并已开始初步研究 检测它们在胚胎发育过程中的表达。我们是 扩大这些研究以分离出迄今为止未知的 一家人。在与L.Williams博士团队的合作下，我们建议使用 用类似的方法获得小鼠成纤维细胞生长因子受体(FGFR)基因。AS 这些不同的基因一旦获得，我们将进行详细的 它们在发育中胚胎中表达的研究。作为关于 这些基因的表达开始积累，应该有可能 推断它们各自在发展中的作用，有些人可能 在发展中的embyo中展示表达模式。作为有关的信息 这些基因的表达开始积累，这应该是可能的 为了推断它们各自在开发中的功能，一些 可能会表现出与发展中的角色一致的表达模式 胚胎心脏或肺的。我们应该得到帮助来画这些 通过与M.Kirschner博士和他的 合作者正在研究b-成纤维细胞生长因子在中胚层诱导中的作用 在青蛙里。 我们的第二组具体目标是获得携带基因突变的小鼠 感兴趣的特定的成纤维细胞生长因子或成纤维细胞生长因子受体基因。这将通过以下方式实现 转基因小鼠胚胎干细胞的体外转染法 对感兴趣的基因进行筛选并选择那些改变了 等位基因已经用同源基因取代了基因的内源拷贝 重组。这样的细胞将被用来产生小鼠品系 改变的基因是杂合的。然后这些小鼠就可以进行杂交了 为目标基因改变产生纯合的后代，并且 评估基因改变的功能后果。结果是 这样的研究应该允许我们评估我们所拥有的任何假说 成纤维细胞生长因子和成纤维细胞生长因子受体基因家族可能功能的研究进展 发展。

Evidence is rapidly accumulating that basic fibroblast growth factor 9b- FGF) and related proteins play an important role in embryonic induction. In mammals, four genes (a-FGF, k-FGF, int-2 and FGF-5) have been identified that have extensive sequence similarity to b-FGF, and more are likely to exist. We are interested in studying the role of these genes in early mammalian development. Our first set of specific goals is to determine when and where each of these genes is expressed in the developing mouse embryo by in situ RNA hybridization and protein localization, and from this information to begin to build a hypothesis about what their function in development might be. However, since the stage- and tissue-specific functions of these different growth factors will depend on the stage- and tissue- specificity of their respective receptors, we also propose to study the expression of FGF-receptor(s) in the developing embryo. In order to achieve these goals, we must have available probes for each of these mouse genes. Thus far, the only mouse gene in the FGF family that has been cloned in int-2. We have employed a variety of techniques that utilize the polymerase chain reaction (PCR) to clone cDNAs for all the known members of the mouse FGF family and have begun to preliminary examination of their expression during embryonic development. We are extending these studies to isolate hitherto unknown members of the family. In collaboration with Dr. L. Williams group, we propose to use similar approaches to obtain the mouse FGF-receptor (FGF-R) genes. As these different genes become available, we will carry out a detailed study of their expression in the developing embryo. As information on the expression of these genes begins to accumulat, it should be possible to infer something about their respective functions in development, some may display patterns of expression in the developing embyo. As information on the expression of these genes begins to accumulate, it should be possible to infer something about their respective functions in development, some may display patterns of expression consistent with a role in development of the embryonic heart or lungs. We should be aided in drawing these inferences by frequent discussions with Dr. M. Kirschner and his collaborators, who are studying the role of b-FGF in mesoderm induction in the frog. Our second set of specific goals is to obtain mice carrying mutations in the specific FGF or FGF-R genes of interest. This will be achieved by transfecting mouse embryonic stem (ES) cells in vitro with altered copies of the gene of interest and selecting those cell in which the altered allele has replaced an endogenous copy of the gene by homologous recombination. Such cells would then be used to generate mouse strains heterozygous for the altered genes. These mice could then be inter-bred to produce offspring homozygous for the targeted genetic change, and the functional consequences of the genetic alterations assessed. The results of such studies should allow us to evaluate any hypotheses we have developed on the possible funcion of the FGF and FGF-R gene familes in development.