Inducible gene expression in the lens using tamoxifen and a GFP reporter

Abstract

In recent years, P1 phage Cre-recombinase has become an indispensable tool for conditional activation and/or inactivation of genes in the mouse. By fusing Cre to a tamoxifen-sensitive form of the estrogen receptor (Cre-ER ™ ) temporal regulation of gene expression can be achieved. Here, we report the initial characterization of the Cre-ER ™ system for controlling gene expression in the lens of the mouse. Cre-ER ™ mice were crossed with a reporter strain (Z/EG; lacZ/EGFP). Following IP injection of tamoxifen into Cre-ER ™ ;Z/EG mice, GFP expression was observed in 1-5% of lens epithelial and fiber cells. GFP expression was maintained for at least six months although, in the fibers, GFP appeared to diffuse from the cells as they were internalized into the lens. The rate of elongation of the fiber cells was determined by measuring the length of GFP-expressing cells at intervals after tamoxifen injection. The results of this calculation suggested that tamoxifen treatment induced GFP expression predominantly in lens epithelial cells. The GFP-positive fibers were apparently derived from this cell population. The strong induced expression of GFP in a small proportion of lens cells allowed individual lens cells to be identified in the intact tissue and should serve as a useful lineage marker to track the fate of lens cells and their descendents. Keywords Lens; Cre recombinase; GFP; inducible expressionThe use of transgenic and gene knockout technologies has greatly enhanced our understanding of the molecular mechanisms underlying lens development. However, the consequences of genetic manipulations often become apparent early in embyrogenesis and persist throughout the remainder of embryonic and adult development. This complicates and occasionally confounds the analysis of the phenotype. To circumvent this difficulty, inducible gene expression systems have been developed that allow the temporal regulation of gene expression in vivo. Here we report the initial characterization of one such system in the mouse lens.Among several strategies for inducible gene expression are those that utilize the Cre-loxP recombination system. This approach takes advantage of the properties of P1 phage Crerecombinase, a 38 kDa enzyme that recognizes a 34 bp DNA sequence called loxP. Introduction

The stratified syncytium of the vertebrate lens

“…Tamoxifen injection resulted in GFP expression by 1-5% of lens epithelial cells and superficial fiber cells. The relatively low level of induction has been attributed to mosaic expression of the Z/EG reporter transgene in the lens (Shi and Bassnett, 2007). For the present purposes, the low induction efficiency was advantageous, because it enabled individual GFP-expressing cells to be visualized in the intact tissue.…”

“…This protocol resulted in GFP expression in the lens within 24 hours. Typically, 1-5% of epithelial cells and young fiber cells were GFP positive (Shi and Bassnett, 2007). For some experiments, lenses from TgN(GFPU)5Nagy mice were used.…”

“…Induced expression of GFP was achieved by crossing Cre-ER TM mice with the Z/EG reporter strain, to generate mice heterozygous for both transgenes, as described (Shi and Bassnett, 2007). Three-week-old Cre-ER TM ;Z/EG animals were given two intraperitoneal injections of tamoxifen (Sigma) dissolved in corn oil (15 mg/ml) to a final dose of 0.15 mg tamoxifen per g body weight.…”

Biological glass: structural determinants of eye lens transparency

“…These projections appear primarily to interconnect cells with their lateral neighbours in adjoining radial cell columns (figures 3d -h and 4a -d ). Early in fibre cell elongation the cells have a smooth, ribbon-shaped form with very small protrusions [13] but, over time, they become increasingly elaborate (figure 4c,d, see also [14]). A seemingly distinct structure, the balland-socket process, is often present on the broad face of differentiating fibre cells, where it connects fibre cells with neighbouring cells located immediately above or below them in a radial cell column (figures 3d and 4c,d ).…”

“…In the adult mouse lens, most fibre cells are incorporated into the syncytial region; only a thin (less than 100 mm) layer of cells at the lens surface is excluded. Shi et al introduced gene induction techniques to the lens field to label individual differentiating fibre cells with GFP in vivo and to follow the fate of the GFP as fibre cells were incorporated into the lens [13]. Curiously, intercellular GFP diffusion was not isotropic (figure 9a -c).…”