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被广泛用于研究基因在发育和成年期的体内功能。标准基因敲除和转基因小鼠已经提供了丰富的信息，但早期胚胎致命性或涉及多个组织的复杂表型往往模糊了主体基因在发育后期、成人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敲除小鼠表现出眼部表型（包括角膜），但胚胎致命性或过早死亡排除了进一步研究这些基因在成年小鼠中的功能的可能性。通过转基因条件表达和组织特异性基因消融获得的小鼠为克服传统转基因和基因靶向小鼠模型的某些局限性提供了一种手段。这可以通过在转基因小鼠中使用组织特异性启动子来驱动Cre和rtTA的表达来实现。例如，已经证明K12角蛋白基因（Krt1.l2）仅在角膜上皮中表达。因此，Krt1.12启动子对于制备表达Cre和rtTA的转基因小鼠是非常理想的，该技术可用于制备角膜上皮特异性基因消融的小鼠品系，并有条件地表达生长因子、受体、转录因子等报告基因。不幸的是，在转基因小鼠中鉴定功能性Krt1.12启动子的尝试被证明是徒劳的。为了克服这一困难，在拟议的研究中，将采用基因靶向技术的敲入策略，分别制备角膜上皮中表达Cre和rtTA的Krtl.12“c”（Specific Aim 1）和Krtl.12+I”TA （Specific Aim 2）小鼠系。这些小鼠系将有助于制备小鼠系，用于研究角膜上皮特异性基因消融和报告基因过表达引起的遗传功能改变。

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.