In vitro comparison studies of truncated rhodopsin promoter fragments from various species in human cell lines

Clin Exp Ophthalmol. 2003 Oct;31(5):445-50. doi: 10.1046/j.1442-9071.2003.00694.x.

Abstract

Background: The aim of the present study was to define the rhodopsin promoter elements of various mammalian species by evaluating the effect of these promoters on transgene specificity and expression in human cell lines. Additional characterization of these promoters was performed in the presence or absence of a human inter-photoreceptor retinoid-binding protein (IRBP) enhancer element.

Methods: Truncated murine, bovine and human rhodopsin promoter fragments containing 1.4 kb of the 5' upstream regulatory sequence were subcloned into pEGFP-N1 expression vector to drive the expression of the green fluorescence protein (GFP). Fluorescence-activated cell sorter analysis was used to determine the strength and specificity of GFP expression in three human cell lines, in the presence or absence of an IRBP enhancer element at the 5' end of the promoters.

Results: It was found that the truncated murine rhodopsin promoter was the strongest in Y-79 retinoblastoma cells but it was less specific than the truncated human rhodopsin promoter. It is surmised that transcription factors located at the 5' proximal region of the rhodopsin promoter fragments were responsible for the expression patterns seen in the present study.

Conclusions: These results indicate that the rhodopsin promoter of choice for in vivo work should be from the same species if specificity is to be maximal and the addition of the IRBP enhancer element could increase transgene expression in photoreceptor cells without deleterious effects on specificity.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Base Sequence / genetics
  • Cattle
  • Cell Line
  • Cell Separation
  • Eye Proteins*
  • Flow Cytometry
  • Gene Expression
  • Green Fluorescent Proteins
  • Humans
  • Indicators and Reagents / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Molecular Sequence Data
  • Peptide Fragments / genetics
  • Promoter Regions, Genetic / genetics*
  • Promoter Regions, Genetic / physiology
  • Retinol-Binding Proteins / genetics
  • Retinol-Binding Proteins / physiology
  • Rhodopsin / genetics*
  • Species Specificity
  • Transcription, Genetic / physiology
  • Transfection
  • Transgenes / genetics

Substances

  • Eye Proteins
  • Indicators and Reagents
  • Luminescent Proteins
  • Peptide Fragments
  • Retinol-Binding Proteins
  • interstitial retinol-binding protein
  • Green Fluorescent Proteins
  • Rhodopsin