Figure 3

Transgenic mouse production and characterization. A, Transgenic strategy used to produce mice that express human E2. LAP-tTA transgenic mice have been previously described [34]. These mice express the tetracycline-controlled transactivator (tTA) from the liver specific LAP promoter. The TRE-E2 transgene contains the tetracycline response element (TRE) as part of the promoter, a synthetic intron (thin line), the human E₂ cDNA, an alanine spacer, a c-myc epitope tag, and SV40 derived polyadenylation sequence. This construct was used to create several lines of transgenic mice. B, Western blot analysis of E2 protein in liver of control and intermediate MSUD mice. Note that the amount of human E2 protein (predicted MW ~54 Kd) in mice from Lines A and 525 A was variable but in many of the animals the amount was similar to the amount of mouse E2 (MW=~47 Kd) in control animals. Re-probing with a c-myc tag antibody confirmed the presence of the transgene derived, c-myc tagged, human E2 in transgenic mice but not in controls. Western blot analysis of brain (C), kidney (D), and muscle (E) revealed negligible amounts of transgene derived E2 in those tissues. All blots were re-probed with an actin antibody to allow amount of protein loaded in each lane to be compared.