| Maintaining apporiated osmotic pressure is essential for the development of lens fiber cells and maintaining lens transparency. Cells respond to osmotic stress by osmoregulation, i.e., regulatory compensation of changes in cell volume, water content, and intracellular solute concentration. Osmoregulation minimizes changes in the concentration of intracellular inorganic ions-in particular Na+ and K+, macromolecules, and metabolites-and is essential for cell metabolism to operate properly. This achieved by adjusting the levels of compatible osmolytes. Compatible osmolytes are small organic solutes, such as glycine betaine or myo-inositol, which are generally accumulated by transporters or enzymes, many of which are transcriptionally regulated. This accumulation occurs through hypertonicity-induced transcription of genes whose promoters are controlled by the enhancer ORE, osmotic response element. These genes include aldose reductase (AR), which mediates the synthesis of the osmolyte, sorbitol, and at least two transporters, the betaine/y-aminobutyric acid transporter (BGT1) and the sodium-myo-inositol cotransporter, which cause cellular accumulation of betaine and inositol, respectively. The cognate transcription factor of the ORE enhancer is OREBP, osmotic response element binding protein. Hypertonicity rapidly causes nuclear translocation and phosphorylation of OREBP and, more slowly, increases OREBP abundance. Hypertonicity-induced binding of the transcription factor OREBP to its cognate DNA element, is associated withincreased transcription for several osmotically regulated genes.Hypertonicity not only induces the transcription of these genes encoding transporters and enzymes for compatible osmolytes through OREBP but also induced some other genes expression like UTA, urea transporter A and HSP70, heat shock protein 70 These suggest the role of OREBP other than osmotic regulation.OREBP mRNA is expressed at low levels in most cells in culture and in virtually all tissues that have been sampled. The low level of expression of OREBP in isotonic cells is reduced further when cells are adapted to growth in hypotonic medium.In addition, OREBP is expressed at much higher levels in embryonic tissues than in adult tissues. OREBP maybe play a role in development.The N terminal of OREBP contains a DNA binding domain and the C terminal of OREBP contains a transactivation domain. Under hypertonic stress, the DNA binding domain cognates with the ORE of those downstream genes, and the transactivation domain actives the transcription. This truncated gene has been shown previously to behave in a dominant-negative manner in cultured cells under hypertonic stress.The experiment described here were designed to inhibit endogenous OREBP activity by expression of the truncated form of OREBP. In order to identify the in vivo function of OREBP in lens, we produced a transgenic mouse expressing the truncated form of OREBP under the control of a murine a A-crystallin promoter to make it specifically in lens. Using this transgenic animal model to study the effect of OREBP in lens development and osmotic regulation.MethodThe initial step of this project was to overexpress truncated form of OREBP specially in mouse lens and it was achieved by introducing multiple copies of the truncated OREBP into mouse genome by transgenic technology. After generating the transgenic mice, the organization of transgene in their genome was charactierized by polymerase chain rection (PCR).Lens development and cataract formation was monitored by slit-lamp microscope. Lenses were further compared under dissection microscope and the size, wet weight and dry weight were measured.To analyze transgene and endougenous OREBP expression level, immunocytochemical analysis of embryonic 13.5, 16.5 and 18.5 days embryonic lens paraffin sections was done using anti-FLAG and anti-endogenous OREBP antibody.Lens aldose reductase mRNA expression level was quantifi...
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