Generation of Vldlr transgenic (Tg) rats. (A) Map of Vldlr genomic DNA and the pCX-Vldlr-IRES-EGFP transgene. Full-length rat Vldlr cDNA was subcloned into the pIRES vector. The Vldlr internal ribosome entry site (IRES) was then subcloned into the pCX-EGFP vector containing the CMV immediate-early enhancer (CMV-IE)/chicken β-actin promoter and enhanced green fluorescent protein (EGFP). Arrows show positions of the PCR primers used to distinguish genomic DNA from the transgene. (B) PCR-based genotyping of transgenic rats. The primers identified a 502-bp wild-type (Wt) genomic DNA fragment and a 417-bp transgene (Tg) fragment. (C) Expression of endogenous (Wt) and transgenic (Tg) Vldlr protein in the liver at P0. Two pups from each line (1 to 3) were used. Vldlr from membrane fractions was detected by Western blotting using an anti-Vldlr antibody (upper panel). The blots were subsequently probed with an anti-β-actin antibody as a protein loading control (lower panel). The results are representative of three independent experiments. (D) qRT-PCR quantification of Vldlr mRNA expression in Wt and Tg whole brain at P0 (lines 1–3). (E) qRT-PCR quantification of Vldlr mRNA expression in Wt and Tg whole brain (upper panel), and prefrontal cortex, hippocampus and cerebellum (lower panels) in adult (2 to 3 months) rats from transgenic line 1. Relative Vldlr expression was obtained by normalizing to Actb from the same cDNA. Results are expressed as a ratio of Wt expression, resulting in a Wt ratio of 1. Error bars represent mean ± SEM (n = 3 to 6 per genotype). * P < 0.05, ** P < 0.01 between Wt and Tg rats.