Figure 1.
Expression of RhoA DN and Rho-K DN by using the Cre-loxP-mediated recombination system. A, Experimental strategy. DBH-Cre transgenic mice were crossed with CAT-RhoA DN or CAT-Rho-K DN mice. In the double-transgenic mice (Cre+/RhoA DN+ or Cre+/Rho-K DN+), Cre-loxP recombination deletes the CAT gene cassette in specific cell types, thereby leading to the expression of the dominant-negative forms. B, Patterns of Cre-loxP recombination. Histological staining of β-gal activity in transverse sections through the thoracic spinal cord of transgenic embryos carrying both DBH-Cre and CAG-CAT-Z transgenes (Cre+/lacZ+) at E9.5–E11.5, showing the staining signals in the developing MN columns in the ventrolateral region (arrow) and in the presumptive progenitor cells in the region around the ventricular zone (arrowheads) of the neural tube. The signals were also observed in the SG and DRG. Scale bar, 50 μm. C, Cre-loxP recombination in developing MNs. Sections through the thoracic spinal cord of the Cre+/lacZ+ double-transgenic embryos at E10.5 were stained with antibodies for Isl1 and β-gal. Isl1-staining signal (red), β-gal-staining signal (green), and their merged image are indicated. As seen in a fourfold-magnified view of the merged image, Isl1 and β-gal signals are localized in the nucleus and the soma, respectively. Thus, double-positive cells show a red nucleus and a green soma in a single cell. Scale bar, 50μm. D, Expression of RhoA DN and Rho-K DN genes. Total RNA prepared from E10.5 embryos was reverse transcribed and analyzed by PCR amplification with CAT, RhoA DN, or Rho-K DN primer set. The size of amplified DNA was 390 bp for CAT, 617 bp for RhoA DN, and 482 bp for Rho-K DN.