Mice Overexpressing rtTA and Cre in Corneal Epithelium

描述（由申请人提供）：小鼠转基因和基因靶向技术已广泛用于研究发育和成年期基因的体内功能。标准的基因敲除和转基因小鼠已经提供了大量的信息，但是早期胚胎致死或涉及多个组织的复杂表型往往掩盖了受试者基因在发育后期、成体或特定组织中的作用。例如，许多转录因子和生长因子及其各自受体的基因敲除小鼠表现出眼部表型（包括角膜），但胚胎致死或过早死亡排除了进一步研究成年人中这些基因功能的可能性。由转基因的条件表达和组织特异性基因切除衍生的小鼠提供了规避常规转基因和基因靶向小鼠模型的某些限制的手段。这可以通过使用组织特异性启动子来驱动Cre和rtTA在转基因小鼠中的表达来实现。例如，已经证明K12角蛋白基因（Krt1.12）仅由角膜上皮表达。因此，Krt1.12启动子将是优秀的转基因小鼠表达Cre和rtTA，一种技术，可用于制备具有角膜上皮特异性基因消融和有条件表达的报告基因，如生长因子，受体，转录因子等的小鼠品系的准备，不幸的是，试图确定一个功能性的Krt1.12启动子在转基因小鼠已被证明是徒劳的。为了克服这一困难，在所提出的研究中，将使用基因靶向技术的敲入策略来制备分别在角膜上皮中表达Cre和rtTA的Krtl.12“c”（Specific Aim 1）和Krtl.12+ 1“TA（Specific Aim 2）小鼠系。这些小鼠品系将是有用的，在准备小鼠品系的研究改变遗传功能的角膜上皮特异性基因消融和报告基因的过表达。

DESCRIPTION (provided by applicant): Techniques of transgenic and gene targeting in mice have been used widely to study in vivo functions of genes during development and adult life. Standard knockout and transgenic mice have been highly informative, but early embryonic lethality or complex phenotypes involving multiple tissues often obscure the roles of subject genes at later stages of development, in adults or in specific tissues. For example, many knockout mice of transcription factors and growth factors and their respective receptors exhibit ocular phenotypes (including the cornea), but embryonic lethality or premature death precludes the possibility of further examining the functions of such genes in adults. Mice derived from conditional expression of transgenes and tissue-specific gene ablation provide a means of circumventing certain limitations of conventional transgenic and gene targeting mouse models. This can be achieved by the use of tissue-specific promoter to drive the expression of Cre and rtTA in transgenic mice. For example, it has been demonstrated that K12 keratin gene (Krt1.l2) is solely expressed by corneal epithelium. Thus, Krt1.12 promoter would be excellent for the preparation of transgenic mice expressing Cre and rtTA, a technique that can be used to prepare mouse lines that have cornea epithelium-specific gene ablation and conditional expression of reporter genes such as growth factors, receptors, transcription factors, etc. Unfortunately, attempts to identify a functional Krt1.12 promoter in transgenic mice have proved fruitless. To circumvent this difficulty, in the proposed studies a knock-in strategy of gene targeting techniques will be used to prepare Krtl.12"c' (Specific Aim 1) and Krtl.12+I"TA (Specific Aim 2) mouse lines expressing Cre and rtTA in corneal epithelium, respectively. These mouse lines will be useful in preparing mouse lines for studies of altered genetic functions from the corneal epithelium-specific gene ablation and overexpression of reporter genes.