TRANSFORMING POTENTIAL OF CELLULAR ONCOGENES

本申请的主要目的是确定是否增加了 三种原癌基因c-mos、c-myc的表达或结构的改变， fos和int-1导致小鼠肿瘤发病率增加。 这些 不适当的时间和不适当的组织影响正常的 发展 一项实验仅关注发展 将点突变引入所谓的 c-mos基因的丝氨酸/苏氨酸激酶编码结构域。 将采用的方法是引入经过修饰的癌基因， 表达或结构到小鼠中利用胚胎来源的干细胞（ES） 细胞 这些细胞用含有以下的质粒构建体转染： 或由诱导型启动子驱动的原癌基因（结构上 改变的原癌基因）。 然后将转染的ES细胞显微注射到 移植到胚泡中以产生嵌合小鼠。 因为胚胎干细胞在胚胎中 具有良好频率的转基因小鼠系，具有修饰的原代- 可以建立致癌基因。 ES细胞/嵌合体系统的两个优点 与DNA显微注射相比， 在嵌合体产生之前表征转染的ES细胞， 通过同源重组靶向突变进入内源性 感兴趣的基因 本申请中描述的实验充分说明了 使用这种基因转移方法来探索原核细胞的潜在作用， 癌基因在肿瘤发生中的作用以及它们在正常 发展 所提出的实验解决了4个具体问题：1） 原癌基因c-mos、c-fos和int-1的表达增加 对小鼠肿瘤的发生和/或正常发育产生不利影响？ 2)是c-fos基因结构的变化， 也被证明是培养细胞中细胞转化所必需的 是小鼠肿瘤形成所必需的吗 3)连接是否升高 小鼠中c-fos和c-mos的表达协同影响发病率， 肿瘤的位置和/或生长 4)组氨酸-190对酪氨酸 c-mos蛋白中取代导致丝氨酸/苏氨酸激酶的丢失 这种点突变会影响生物学功能吗？

The main objective of this application is to determine whether increased expression or change in the structures of three proto-oncogenes, c-mos, c- fos, and int-1, results in an increased incidence of tumors in mice. These inappropriate times and in inappropriate tissues affects normal development. One experiment focuses solely on the developmental consequences of introducing a point mutation into the purported serine/threonine kinase encoding domain of the c-mos gene. The approach that will be taken to introduce oncogenes having modified expression or structure into mice makes use of embryo-derive stem (ES) cells. These cells are transfected with plasmid constructs containing either or proto-oncogenes driven by an inducible promoter (structurally altered proto-oncogenes). Transfected ES cells are then microinjected in to blastocysts to generate chimeric mice. Since ES cells populate the germ line with good frequency, transgenic mouse lines having modified proto- oncogenes can be established. Two advantages of the ES cell/chimera system as compared to DNA microinjection to produce transgenics are the ability to characterize transfected ES cells prior to chimera production and the ability to target mutations by homologous recombination into endogenous genes of interest. The experiments described in this application make full use of this method of gene transfer to explore the potential role of proto- oncogenes in tumorigenesis as well as their functions during normal development. The proposed experiments address 4 specific questions: 1) Does the elevated expression of the proto-oncogenes c-mos, c-fos, and int-1 increase the incidence of tumors in mice and/or affect normal development adversely? 2) Are changes in the structure of the c-fos gene that have been demonstrated to be necessary for cell transformation in cultured cells also necessary for tumor formation in mice? 3) Does the join elevated expression of c-fos and c-mos in mice synergistically affect the incidence, location, and/or growth of tumors? 4) Does a histidine-190 to tyrosine substitution in c-mos protein result in loss of serine/threonine kinase activity and does this point mutation affects biological function?