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.