Establishment Of Rod Opsin Promoter-green Fluorescent Protein Transgenic Mice

The retina and the central nervous system are both developed from the neural tube. Retinas are genuine parts of the brain and the retinal development study can act as a model of the central nervous system development. The retina disorder is a vital part of blind-caused disease. Point mutations in some gene in photoreceptor cells have been implicated as causative of some form of such retina disease as retinal degeneration retinitis pigmentoma. The study of retina development,especially the photoreceptor development,will help to understand those diseases. Before the development procession of retina was further studied,an animal model with retina labeled should be made. Transgenic mice have many advantages as this subject. The structure and functions of the mice genomic genes are similar with that of human being. Rapid development and low feeding cost are also considered. Transgenic mice described in this paper should have used beyond the animal model for retinal development. Transplanting the retina expressing GFP to the retina without fluorescent,we will conveniently judge whether the exogenous retina is alive by the GFP expressing pattern and make traces of the migrating or penetrating way of the cells of both the host and the implant.'In previous studies,a 4.4kb promoter sequence was used to direct the 3 -galactosyltransferase ( LacZ ) gene retina-specific express in transgenic mouse lines. (Zack DJ,et al. 1991). The innovations of our paper exist in two aspects:2.1kb promoter sequences instead of 4.4kb sequences and GFP-labeling instead of LacZ-labeling. Shorter promoter sequence help to understand the regulatory element more precisely and GFP-labeling makes observation more easily. In our research,we try to label the retina of a kind of mammalian-mouse by GFP,whereas previously analogous studies were limited to someamphibian and fish.The rod opsin promoter (ROP) obtained from the murine genome by polymerase chain reaction was cloned into PGEM-T vector,and was named ROP-T. The Nde I / Xba I fragment of ROP-T was ligated into the Nhe I / Vsp I fragment of pEGFP-Cl(Clontech). The constructed expression vector,named ROP-GFP,was linearized by Not I,and diluted into 2ug/ml and injected into the pronuclei of fertilized one-cell eggs. Microinjected eggs were incubated overnight at 37 D,5% CO2 in Ml6 media and allowed to develop to the two-cell stage. Surviving two-cell stage eggs were transplanted into the oviducts of pseudopregnant foster mothers. Transgenic animals were identified by polymerase chain reaction analysis of genomic DNA prepared from tail samples. After the PCR-positive mice have generated posterities,the eye balls were excised and used to make freezing section. Photos were taken under the fluorescent microscope. The offspring of the Founder mice were identified by PCR.There were 3 positive Founder mice and 9 positive offspring out of 12 babies by PCR. The retina showed strong fluorescent expression.The results showed that the 2.1 kb promoter sequences contained the entire element directing exogenous gene expression in retina.Replacing the marker gene with other exogenous genes,we will use ROP to direct other exogenous genes to express in the retina. The technica described in our experiment provided a feasible consideration to repair some birth defect of retina by gene therapy. The fact that point mutation of the rod opsin is one of the pathogeny makes our consideration more attractive.