In vitro differentiated adipocytes from a Foxc2 reporter knock-in mouse as screening tool.

• We have developed a generic model for in vitro high-throughput screening for agents regulating transcription of genes in the mouse genome here exemplified by Foxc2, a forkhead transcription factor involved in regulation of adipocyte metabolism. We made a Foxc2-LacZ reporter "knock-in" mouse in which one of the two Foxc2 alleles has been inactivated and replaced by a LacZ reporter gene. Mouse embryonic fibroblasts, derived from such mice, were differentiated in vitro to adipocytes and used in cell-based screens. Forskolin as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) increased levels of Foxc2nLacZ fusion protein. We could also demonstrate that this was paralleled by an increase in Foxc2 mRNA, transcribed from the wild type allele. This generic method offers a novel way of identifying both positive and negative upstream regulators of a gene, using high-throughput screening methodology. In a cell-based screen using such methodology we demonstrate efficacy by identifying NKH477 as a Foxc2 activating compound.

Nyckelord

Adipocytes

cytology

metabolism

Animals

Cell Differentiation

drug effects

Cells

Cultured

Drug Evaluation

Preclinical

Embryo

Mammalian

cytology

Female

Fibroblasts

cytology

metabolism

Forkhead Transcription Factors

genetics

Gene Knock-In Techniques

Lac Operon

Male

Mice

Small Molecule Libraries

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)