An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo

Am J Physiol Renal Physiol. 2005 Aug;289(2):F481-8. doi: 10.1152/ajprenal.00332.2004. Epub 2005 Mar 22.

Abstract

The utility of promoter fragments isolated from the 5'-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (beta-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas beta-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (beta-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39-42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.

Publication types

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

MeSH terms

  • Animals
  • Cell Line
  • Gene Expression Regulation / physiology*
  • Genetic Techniques*
  • Genotype
  • Green Fluorescent Proteins / metabolism
  • Integrases / biosynthesis
  • Integrases / genetics
  • Kidney / cytology*
  • Kidney / metabolism*
  • Mice
  • Mice, Transgenic
  • Plasmids / genetics
  • Promoter Regions, Genetic
  • Transgenes / genetics*
  • beta-Galactosidase / metabolism

Substances

  • Green Fluorescent Proteins
  • Cre recombinase
  • Integrases
  • beta-Galactosidase