Delivery of genes and fluorescent dyes into cells of the intact lens by particle bombardment.
The authors report the use of a particle bombardment technique to deliver exogenous genes and fluorescent dyes into living fibre cells in the intact lens. Gold particles were coated with plasmid DNA encoding green fluorescent protein (GFP) or the lipophilic fluorescent probes DiI and DiO. The particles were introduced into embryonic chicken or neonatal mouse lenses using a pressurized helium charge. A significant fraction of particles penetrated the capsule and came to rest in the superficial lens cortex. Over the range tested, varying the particle size or the pressure of the helium charge had little effect on the final distribution of particles within the lens. Particle bombardment was used initially to deliver DiI and DiO into the lens. Within a few hours, bombarded lenses exhibited multicolored membranous labelling of individual, elongating fibre cells. The particle bombardment technique was also used to introduce a plasmid encoding GFP. After overnight incubation, many fibre cells in the bow region expressed GFP. On close examination by confocal reflectance microscopy, each expressing cell was found to contain a gold particle lodged in its nucleus. The authors examined the fate of GFP-expressing fibre cells over a period of 1 week in organ culture. In the embryonic chicken lens, transfected fibres showed modest (approximately two-fold) elongation. In contrast, GFP-expressing mouse lens fibres underwent dramatic elongation, reaching the anterior and posterior sutures after 7 days in culture. These species differences may reflect the fact that mitosis continued at a near normal rate in the cultured mouse lens but declined precipitously in the cultured chicken lens. These results suggest that particle bombardment, in conjunction with appropriate cell culture conditions, may prove useful in visualizing the behaviour of differentiating fibre cells in the living intact lens in vitro.
Shestopalov
Valery I
VI
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO, 63110, USA.
Missey
Heather
H
Bassnett
Steven
S
D000818:Animals; D019470:Biolistics / Q000379:methods*; D002454:Cell Differentiation; D002642:Chick Embryo; D005456:Fluorescent Dyes / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins; D007908:Lens, Crystalline / Q000166:cytology* / Q000196:embryology / Q000254:growth & development; D008164:Luminescent Proteins / Q000008:administration & dosage / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009924:Organ Culture Techniques; D013045:Species Specificity
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins