Quantification of EGFP and Arrestin-EGFP in Transgenic Xenopus Rods

Abstract

Abstract: : Purpose: To quantify the expression of EGFP and Arrestin-EGFP in living transgenic CHO cells and Xenopus rod cells. Methods: We have constructed a 3D confocal scanning laser microscope (CLSM) whose point-spread function has a volume of about 0.l µm³ and used this scope to measure protein expression levels in living cells. A line of stably expressing CHO cells (pDP3) was created with lipofectamine transfection of EGFP gene under control of a CMV promoter. Transgenic Xenopus tadpoles were made by restriction enzyme mediated incorporation into eggs of linearized DNA which included a Xenopus opsin promoter followed by the EGFP gene or an Arrestin-EGFP fusion construct. Quantitative Western blotting was used to determine the average expression level in populations of cells counted with cytometric methods. The CLSM was calibrated as a quantitative fluorimeter using spectrophotometrically measured concentrations of recombinant EGFP in a microchamber. Results: As measured with the CLSM, pDP3 cells had an average envelope volume of ~ 1 pl and expressed EGFP at an average concentration of 5.6 ± 0.3 µM (mean ± 95% c.i., n = 273). Western analysis (6 experiments) yielded a population average expression of 7 (± 4) × 10^–18 mols EGFP/cell, predicting an average concentration of about 7 µM, close to that estimated by CLSM. The measurements with pDP3 cells confirm the general validity of the approach. Rod cells expressing EGFP had up to 15 µM protein locally, but the fluorescence intensity varied up to 4-fold within individual rods (the outer segment being 2- to 3-fold less intense than the brightest voxels in the inner), and by more than 10-fold between rods from the same retina. The latter variation had a mosaic character, and we used CLSM analysis of tangential frozen sections to quantify the mosaicism. In the dark, rods expressing Arrestin-EGFP exhibited a distribution of fluorescence similar to that of EGFP, but the fluorescence distribution shifted very strongly to the outer segment upon rhodopsin bleaching. Conclusions: The local concentrations of EGFP and EGFP fusion proteins can be quantified in living transgenic Xenopus rods and used to examine cytoplasmic compartments and redistribution of proteins upon stimulation.

Keywords: photoreceptors • microscopy: light/fluorescence/immunohistochem • protein structure/function