Exogenous gene expression and protein targeting in lens fiber cells.
OBJECTIVE
To test the ability of lens fiber cells at various stages of differentiation 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-cultured embryonic chicken lenses. Protein expression was visualized by confocal microscopy.


RESULTS
GFP expression was detected within 12 hours of microinjection, evenly distributed throughout the cytoplasm of the injected cell. All 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 fusion protein was synthesized intact and properly inserted in the fiber cell plasma membrane. In contrast, N-terminal fusions were cleaved during synthesis, resulting in retention of the GFP tag in the endoplasmic reticulum.


CONCLUSIONS
Microinjection of expression plasmids is an effective technique for introducing exogenous genes into individual fiber cells. With this approach, the results show that fiber cells are transcriptionally and translationally competent until the time of organelle loss, and that fiber cells deep within the lens are capable of synthesizing new plasma membrane proteins. The techniques described here should have broad application in studies of fiber cell differentiation and provide a useful complement to conventional transgenic approaches.
Shestopalov
V I
VI
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Bassnett
S
S
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis* / Q000235:genetics; D015153:Blotting, Western; D002454:Cell Differentiation; D002642:Chick Embryo; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007908:Lens, Crystalline / Q000196:embryology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D008845:Microinjections; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D015703:Antigens, CD; D008164:Luminescent Proteins; D051916:Membrane Cofactor Protein; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins