Novel Technologies For Skin-Specific Gene Expression

描述(由申请人提供)： 这笔赠款的目标是开发两项新技术；一项是用于在小鼠中控制转基因表达的空间和时间；另一项是用于产生转基因小鼠。在小鼠体内导入和表达转基因有几个困难。其中包括不可预测的插入位点(S)、不可预测的拷贝数和不可预测的表达。将开发的第一项技术将使用噬菌体RNA聚合酶(RNAP)以时间和空间方式调节转基因表达。RNAP与雌激素或孕激素配体结合区融合，在没有配体的情况下定位于细胞质，而在添加配体时转移到细胞核。因此，只有当RNAP定位于细胞核时，才能表达受RNAP启动子转录控制的cDNA。时间控制通过应用配基实现，空间控制通过将杂交的RNAP/配体结合结构域蛋白置于组织特异性启动子的控制下实现。第二种技术使用改进的基因打靶方法，允许将cDNA插入到预定的基因中，从而使内源启动子控制cDNAs的表达。将描述产生工程化胚胎干细胞以产生嵌合体小鼠所需的选择方法。皮肤是发展这些技术的理想器官。首先，有许多特征明确的皮肤特异性促进剂可供选择。其次，皮肤是可接触的，可以在不进行安乐死或手术的情况下轻松采集组织样本，还可以进行表型变化的目测检查。此外，在基因治疗过程中，皮肤通常被认为是引入凝血因子等基因的场所。因此，这些程序，如果付诸实践，可以作为一种快速和相对容易的方法来确定基因治疗应用的原则证明。

DESCRIPTION (provided by applicant): The goal of this grant is to develop two new techniques; one is for the spatial and temporal control of transgene expression in mice; and the other for generation of transgenic mice. There are several difficulties with introducing, and expressing a transgene in mice. These include unpredictable insertion site(s), unpredictable copy number, and unpredictable expression. The first technique to be developed will use a bacteriophage RNA polymerase (RNAP) to regulate transgene expression in a temporal and spatial manner. The RNAP is fused to an estrogen or progesterone ligand-binding domain such that it will be localized to the cytoplasm in the absence of ligand but translocate to the nucleus when ligand is added. Thus, a cDNA under transcriptional control of the RNAP promoter can only be expressed when the RNAP localizes to the nucleus. Temporal control is achieved by application of the ligand and spatial control is achieved by placing the hybrid RNAP/ligand binding domain protein under control of a tissue-specific promoter. The second technique uses modified gene-targeting approaches to allow the insertion of a cDNA into a predetermined gene such that the endogenous promoter will control cDNA expression. Described will be the selection methods needed to generate the engineered embryonic stem cells to generate the chimeric mice. The skin is an ideal organ in which to develop these techniques. First, there are a number of well- characterized cutaneous specific promoters available. Secondly, the skin is accessible, permitting easy tissue sampling without euthanasia or surgery and also visual inspection for phenotypic changes. Further, the skin is often considered as a site for the introduction of genes, such as clotting factors, in gene-therapy procedures. Thus these procedures, if rendered into practice, could serve as a quick and relatively easy method to determine proof-of-principle for gene therapy applications.