Differential Expression Of Embryonic Stem Cell Genes In The Central And Peripheral Lens Epithelial Cells

Lens epithelial cells (LECs) divide continuously throughout the life-span, and their daughter cells migrate from the germinative zone to the equatorial region, elongate and finally differentiate into lens fiber cells. Our previous studies suggest the existence of lens stems cell in the central epithelium of rabbit lens. One of the purposes of the present study is to confirm our previous conclusion. To do so, I have isolated the lens epithelial cells from both central and periphery region of rabbit lenses of 6-months and cultured them in the in vitro conditions. The cultured cells were subjected to analysis by Reverse transcription-linked polymerase chain reaction (RT-PCR) and immunocytochemistry, and following results were obtained:1) lens epithelial cells from different regions can be successfully cultured in vitro; 2) Both RT-PCR and immunocytochemistry analysis revealed that the embryonic stem cell genes CD9 and SOX2 were expressed much stronger in the primary cultures derived from the central lens epithelium than those from the periphery lens epithelium. These results provide further support to the conclusion that the central lens epithelium contains lens stem cells.It is well established that lens differentiation occurs through a process of self removal of the lens cellular organelles. During this process, one of the most important components is the DNaseâ…¡-like acidic DNase (DLAD). The gene coding for this protein has been previously cloned. However, the study on its function has been hampered due to the lack of an antibody recognizing this protein. To explore the functional role of DLAD in the lens differentiation, another part of my thesis research was to produce an antibody against DLAD. To do so, I first generated and purified the GST fusion protein with the catalytic subunit of PP-2A, an important protein serine/threonine phosphatases in vertebrates eye and other tissues that have been actively studied in our laboratory. Using this protein as antigen, I successfully produced antiserum against PP-2Ac which recognizes the endogenous PP-2A in retinal pigment epithelial cells.After this positive experience on antibody production, I started to prepare the antibody against DLAD. Unfortunately, in my studies, I found that production of the DLAD protein in bacterial system was practically unsuccessful. The conclusion from this study is that mouse DLAD cDNA may not be easily expressed in prokaryotic cells. Part of the reasons may be due to the fact the cDNA encodes a DNase which potentially can degrade the host DNA and thus its production is inhibited by the host system in an unknown mechanism.