Generation of transgenic rats.

<p>The transgene construct, pTet-shIR (A), contains two expression cassettes: One expresses shRNA against the insulin receptor (shIR) under the control of the human H1 promoter carrying a tetracycline operator (tetO) sequence. The second cassette consists of a tetracycline repressor (tetR) cDNA followed by a polyadenylation site (pA) and is driven by the CAGGS promoter. An RNase protection assay (RPA) probe was designed to bind to the loop and antisense strand of the hairpin. Primers TetRfor and TetRrev (arrowheads) were used for genotyping of rats. (B) Expression of the shRNA was detected by RPA in 20 µg of total RNA isolated from white adipose tissue (WAT) of wild-type (WT) and transgenic (Tet14 and Tet29) rats treated with doxycycline (DOX, 2 mg/mL) for 4 days. M: RNA Decade marker; Y: yeast RNA; Y-: yeast RNA without RNase digestion; nt: nucleotides. (C) Expression of insulin receptor (IR), tetracycline repressor (tetR), and ß-actin were detected by Western blot in 20 µg of WAT, brain and heart protein from the same rats.