Temporally and spatially controllable gene expression and knockout in mouse urothelium

Am J Physiol Renal Physiol. 2010 Aug;299(2):F387-95. doi: 10.1152/ajprenal.00185.2010. Epub 2010 Apr 28.

Abstract

Urothelium that lines almost the entire urinary tract performs important functions and is prone to assaults by urinary microbials, metabolites, and carcinogens. To improve our understanding of urothelial physiology and disease pathogenesis, we sought to develop two novel transgenic systems, one that would allow inducible and urothelium-specific gene expression, and another that would allow inducible and urothelium-specific knockout. Toward this end, we combined the ability of the mouse uroplakin II promoter (mUPII) to drive urothelium-specific gene expression with a versatile tetracycline-mediated inducible system. We found that, when constructed under the control of mUPII, only a modified, reverse tetracycline trans-activator (rtTA-M2), but not its original version (rtTA), could efficiently trans-activate reporter gene expression in mouse urothelium on doxycycline (Dox) induction. The mUPII/rtTA-M2-inducible system retained its strict urothelial specificity, had no background activity in the absence of Dox, and responded rapidly to Dox administration. Using a reporter gene whose expression was secondarily controlled by histone remodeling, we were able to identify, colocalize with 5-bromo-2-deoxyuridine incorporation, and semiquantify newly divided urothelial cells. Finally, we established that, when combined with a Cre recombinase under the control of the tetracycline operon, the mUPII-driven rtTA-M2 could inducibly inactivate any gene of interest in mouse urothelium. The establishment of these two new transgenic mouse systems enables the manipulation of gene expression and/or inactivation in adult mouse urothelium at any given time, thus minimizing potential compensatory effects due to gene overexpression or loss and allowing more accurate modeling of urothelial diseases than previously reported constitutive systems.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Cell Proliferation
  • Chromatin Assembly and Disassembly / drug effects
  • Doxycycline / pharmacology*
  • Feasibility Studies
  • Gene Expression Regulation / drug effects*
  • Gene Knockout Techniques*
  • Genes, Reporter
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Histones / metabolism
  • Integrases / genetics
  • Lac Operon
  • Membrane Proteins / genetics
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Promoter Regions, Genetic
  • Protamines / genetics
  • RNA, Messenger / biosynthesis*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Time Factors
  • Transcription, Genetic / drug effects
  • Transcriptional Activation / drug effects
  • Uroplakin II
  • Urothelium / drug effects*
  • Urothelium / metabolism

Substances

  • Histones
  • Membrane Proteins
  • Prm1 protein, mouse
  • Protamines
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • Upk2 protein, mouse
  • Uroplakin II
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Cre recombinase
  • Integrases
  • Doxycycline