HOMEOBOX GENES AND THE MOLECULAR CONTROL OF LIMB PATTERN

我们感兴趣的是了解空间形态的遗传控制 在有机体的发育过程中。 有一类基因 在控制发育系统中模式中显示出重要的是 那些含有同源异型盒的基因。 该项目的长期目标是 专注于一个简单结构的发展，再生蝾螈肢体， 来鉴定所有参与这一过程的同源盒基因 并阐明其在调节中的作用。 这些角色可以 包括指定特定肢体（前肢或 后肢）或确定基本肢体轴（近端/远端， 背侧/腹侧和前/后）。 一些基因与 果蝇的Antp类同源异型盒已经被克隆， 严格杂交 两个是前肢特有的，一个是 后肢 其他类含有同源框的基因将被分离出来 使用一组简并寡核苷酸。 其他职能相关 基因已通过差减文库筛选进行鉴定。 这些cDNA 然后将克隆用于分离基因组序列。 两者都将 映射。 将通过北方印迹进一步表征克隆， 原位杂交以确定其时空表达 模式. 使用不同的实验操作，视黄酸可以 用来重新编程每个再生肢体轴。 表达模式 的克隆基因，因此将进行研究后，不同的维甲酸 酸处理，以测试它们在轴规范中的可能作用。 再生的细胞可以转移到组织培养中，诱导摄取 通过脂质体转染或利用VSV-假型逆转录病毒载体， 然后重新引入肢体，同时保留 参与再生。 这项技术将被用于错误表达 同源框cDNA克隆在不适当的空间和时间位置。 同源重组技术将被开发出来， to be inactivated灭活as well. 体外基因转移后RNA酶 保护将测试这些基因是否相互反式调节 表情 将基因组克隆的启动子区连接到标记上 基因如lac Z（编码β-半乳糖苷酶）将使 决定基因特异性的顺式作用元件的解剖 表达模式 这种模式可能部分取决于血统 关系。 再生克隆的谱系将通过 用VSV假型逆转录病毒载体感染再生肢体 携带lac Z，并对β-半乳糖苷酶进行组织学染色， 包含后期的克隆。

We are interested in understanding the genetic control of spatial form during the development of an organism. One class of genes that have been shown to be important in controlling pattern in developmental systems are those containing homeoboxes. The long term goal of this project is to focus on the development of a simple structure, the regenerating newt limb, to identify all of the homeobox containing genes involved in that process and to elucidate the role each plays in its regulation. such roles may include specifying the unique morphology of a particular limb (forelimb or hindlimb) or determining the basic limb axes (proximal/distal, dorsal/ventral and anterior/posterior). Several genes related to the drosophila Antp class of homeobox have already been cloned by low stringency hybridization. Two are specific to the forelimb, one to the hindlimb. Other classes of homeobox-containing genes will be isolated using a set of degenerate oligonucleotides. Other functionally related genes have been identified by a subtractive library screen. These cDNA clones will then be used to isolate genomic sequences. Both will be mapped. The clones will be further characterized by northern blots and in situ hybridization to determine their temporal and spatial expression patterns. Using different experimental manipulations, retinoic acid can be used to reprogram each of the regenerating limb axes. Expression patterns of the cloned genes will therefore be investigated after different retinoic acid treatments to test their possible roles in axis specification. Regenerating cells can be transferred to tissue culture, induced to take up DNA either by lipofection or utilizing VSV-pseudotyped retroviral vectors, and then reintroduced into the limb while retaining the capacity for participating in regeneration. This technology will be used to misexpress the homeobox cDNA clones in inappropriate spatial and temporal locations. Homologous recombination techniques will be developed to allow these genes to be inactivated as well. In vitro gene transfer followed by RNAse protection will test whether these genes trans-regulate each others expression. Attaching the promotor regions of genomic clones to marker genes such as lac Z (encoding beta-galactosidase) will enable the dissection of cis-acting elements that determine the genes specific expression patterns. Such patterns may in part be determined by lineage relationships. Lineages of regenerating clones will be investigated by infecting regenerating limbs with VSV-pseudotyped retroviral vectors carrying lac Z, and histologically staining for beta-galactosidase- containing clones at later stages.