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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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

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.[1]


  1. An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo. Moeller, M.J., Soofi, A., Sanden, S., Floege, J., Kriz, W., Holzman, L.B. Am. J. Physiol. Renal Physiol. (2005) [Pubmed]
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