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.
Moeller
Marcus J
MJ
Institute for Anatomy and Cell Biology, University of Heidelberg, Germany. marcus.moeller@urz.uni-heidelberg.de
Soofi
Abdulsalam
A
Sanden
Silja
S
Floege
Jürgen
J
Kriz
Wilhelm
W
Holzman
Lawrence B
LB
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000502:physiology*; D005821:Genetic Techniques; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D019076:Transgenes / Q000235:genetics*; D001616:beta-Galactosidase / Q000378:metabolism
D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases; D001616:beta-Galactosidase