Novel Technologies For Skin-Specific Gene Expression

描述（由申请人提供）： 这项资助的目标是开发两种新技术;一种是用于小鼠转基因表达的空间和时间控制;另一种是用于转基因小鼠的产生。在小鼠中引入和表达转基因有几个困难。这些包括不可预测的插入位点、不可预测的拷贝数和不可预测的表达。第一项技术将使用噬菌体RNA聚合酶（RNAP）以时空方式调节转基因表达。 RNAP与雌激素或孕酮配体结合结构域融合，使得其在不存在配体的情况下定位于细胞质，但在添加配体时易位至细胞核。因此，在RNAP启动子的转录控制下的cDNA只能在RNAP定位于细胞核时表达。通过应用配体实现时间控制，通过将杂合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空间控制。第二种技术使用改良的基因靶向方法，以允许将cDNA插入预定基因中，使得内源启动子将控制cDNA表达。将描述产生工程化胚胎干细胞以产生嵌合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，有许多充分表征的皮肤特异性启动子可用。其次，皮肤是可接近的，允许容易的组织取样而无需安乐死或手术，并且还可以目视检查表型变化。此外，在基因治疗过程中，皮肤通常被认为是引入基因（如凝血因子）的位点。因此，这些程序，如果付诸实践，可以作为一个快速和相对容易的方法来确定基因治疗应用的原理证明。

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.