目的：利用Cre/LoxP系统建立眼组织特异性Smad4基因敲除小鼠并进行初步表型分析。 方法：通过PAX6启动子驱动的Cre转基因小鼠（ Le-Cre ）作为介导敲除的工具鼠，将其与 Smad4条件基因小鼠（Smad4fl/fl）结合获得Le-Cre特异性Smad4基因敲除小鼠或变异小鼠（ Le-Cre；Smad4fl/fl）。为了证实条件性基因敲除的特异性及有效性，通过PCR技术对小鼠或鼠胚进行了相关基因分析，特定组织绿色荧光蛋白（ GFP）的表达及Smad4蛋白的检测，同时利用ROSA26报导基因小鼠藉LacZ染色显示Le-Cre在眼组织的表达；并对Smad4变异小鼠的表型进行了观察。 结果：早在E10．0左右，鼠胚晶状体位置及眼周外胚层发现强荧光的GFP表达，表明Le-Cre明确出现在特定的靶组织。通过小鼠基因型分析，确定了小鼠或鼠胚是否携带Cre重组酶基因、Smad4条件性等位基因和/或Rosa报导基因，对小鼠晶体DNA的基因分析也证实Smad4基因在某些特异性眼组织内得到剔除。通过在Cre转基因小鼠导入报导基因，并籍LacZ染色对Cre重组酶的表达进行检测，显示了Cre重组酶的时空表达及其表达的组织特异性。 Smad4免疫组化染色显示，在正常发育胚鼠眼可见广泛的Smad4表达，早期主要存在于胞浆，随着胚眼发育向核内转移；而在Smad4变异鼠胚眼，结果表明 Smad4在Cre重组酶所表达的组织内缺乏表达。观察发现Smad4变异鼠可以顺利存活，但表现出眼球缩小、眼窝凹陷、眼睑畸形开放、闭合不能及眼周毛发脱失的外观。 结论：通过基因和蛋白水平的检测证实了Le-Cre；Smad4基因敲除鼠的建立及Smad4在变异小鼠中表达的缺乏。Smad4在眼组织的敲除导致了眼及附属器的明显异常，为深入了解眼球发育及Smad4对其影响的机制提供了一个可信的动物模型。%AIM: To make further phenotypic analysis by establishing the mouse model of specific Smad4 conditional gene knockout in ocular tissue by Cre/LoxP system of this kind of mouse model. METHODS: Mouse of specific Smad4 conditional knockout in lens ectoderm ( Le-Cre; Samd4fl/fl or also called mutant mouse) was obtained by mating the Pax6 promoter-driven Cre transgenic mouse ( Le-Cre ) with Smad4 wildtype mouse ( Smad4 fl/fl).To confirm that Smad4 has been conditionally inactivated only in the specific tissue of ectoderm such as lens, cornea and ectoderm of the eyelids so on.A series of assays were carried out to reveal the validity and specificity of Smad4 gene knockout at molecular and cellular levels, including genotyping by PCR, detection of green fluorescence protein ( GFP) in specific tissue and Smad4 protein.The expression of Le-Cre from Lacz staining using ROSA26 reporter genes in specific ocular tissue of mice can be visualized.Preliminary phenotype of mutant mouse was also observed. RESULTS: As early as around E10.0, strong GFP expression was observed in the embryonic lens and periorbital ectoderm of the mice, which showed Le-Cre was expressed in specific target tissue. Through genotyping for Smad4, Cre and Rosa genes, the mice were determined if they have carried Cre, Smad4 allele or Rosa reporter gene.It was further confirmed by lens-sampled genotyping that Smad4 gene was removed from some specific tissue such as lens.The spatial-temporal expression and tissue specificity of Le-Cre recombinase was also revealed by LacZ staining of Rosa; Le-Cre double transgenic mouse. According to Immunohistochemical staining, Smad4 was widely expressed in normal embryonic eyes, mainly appearing in the cytoplasm at the early embryonic stage and were transferred to nucleus with gestation developing, while in mutant embryonic eyes, Smad4 was void of expression in Cre-expressed tissues. It was observed that Smad4 mutant mouse could survive the conditional gene knockout.But those mice showed abnormal appearance such as microphthalmia, sunken socket, abnormal eyelid opening, and eyelid closure failure and periocular hair loss. CONCLUSION: In this study, mouse model of Smad 4 conditional knockout is precisely established and lack of expression of Smad4 in mutant mice is confirmed by related genetic and proteic detection. Lack of Smad4 expression in specific ocular tissues of mutant mouse can result in the abnormality of eye and adnexa, which provides a reliable animal model to investigate the ocular development and the roles of Smad4 on it.
展开▼
