Exogenous gene expression and protein targeting in lens fiber cells

PURPOSE. To test the ability of lens fiber cells at various stages of diffe rentiation to transcribe and translate microinjected DNA templates. METHODS. Expression plasmids encoding green fluorescent protein (GFP) or a GFP-tagged membrane protein (human CD46) were microinjected into organ-cult ured embryonic chicken lenses. Protein expression was visualized by confoca l microscopy. RESULTS. GFP expression was detected within 12 hours of microinjection, eve nly distributed throughout the cytoplasm of the injected cell. AU nucleated fiber cells were competent to express GFP, whereas the anucleated central fiber cells were not. When GFP was fused to the C-terminal of CD46, the fus ion protein was synthesized intact and properly inserted in the fiber cell plasma membrane. In contrast, N-terminal fusions were cleaved during synthe sis, resulting in retention of the GFP tag in the endoplasmic reticulum. CONCLUSIONS. Microinjection of expression plasmids is an effective techniqu e for introducing exogenous genes into individual fiber cells. With this ap proach, the results show that fiber cells are transcriptionally and transla tionally competent until the time of organelle loss, and that fiber cells d eep within the lens are capable of synthesizing new plasma membrane protein s. The techniques described here should have broad application in studies o f fiber cell differentiation and provide a useful complement to conventiona l transgenic approaches.