Fig. 2. Efficiency of recombination in JoP6 mice. (A) Genomic PCR was performed with the primers JoP6F and JoP6R on DNA isolated from the pJoP6 plasmid and cortices from wild-type (negative control), JoP6 and JoP6;Emx1^IREScre embryos. The PCR amplifies a 1831 bp fragment from unrecombined DNA templates and from pJoP6 plasmid and JoP6 cortex, whereas an additional 261 bp DNA band is seen with the DNA sample from the JoP6;Emx1^IREScre cortex. (B,B') Abundant GFP fluorescence is detected on coronal sections of E14.5 JoP6 cortex, whereas the majority of the cells in the VZ and SVZ of the JoP6;Emx1^IREScre cortex are GFP-negative. Arrows indicate preserved GFP⁺ cells in the VZ. Note the preserved fluorescence in the thin CP and the striatum (str). (C) Whole-mount staining for ß-gal indicates specific expression of lacZ in the telencephalon (tel), olfactory bulb (ob) and scattered cells in the diencephalon (di) of E15.5 JoP6;Emx1^IREScre cortex (bottom), whereas the mesencephalon (mes) and the metencephalon (mt), as well as the JoP6 control brain (top), are unstained. (D) Western blot confirms higher level of Pax6 in the E15.5 JoP6;Nex-Cre as compared with the JoP6 cortex. (E) E12.5 section of JoP6;Emx1^IREScre cortex reveals weak ß-gal staining of individual or aggregated (arrows and inset) progenitors. (F) At E15.5, ß-gal⁺ cells are within LP/VP, IZ and the lower part of the CP. (G) Strong ß-gal staining is seen at E18.5 in late progenitors as well as postmitotic cells of the CP and (H) at P28 throughout the whole cortex. (I,I') Pax6 immunohistochemistry on sections of E18.5 JoP6 (I) and JoP6;Emx1^IREScre (I') cortex, showing ectopic Pax6 expression in postmitotic cells in the IZ and the lower part of the CP. The magnifications of the framed area in I' demonstrate Hoechst and Pax6 co-labeling. (J,K) q-PCRs with RNA isolated from cortex of JoP6 control, JoP6;Emx1^IREScre, and JoP6;Nex-Cre normalized to 18S RNA indicate upregulation of Pax6 in the double-transgenic cortices (J) and specific elevation of the level of Pax6 transcripts in the JoP6;Emx1^IREScre cortex, whereas the level of Pax6-5a is in fact diminished, as compared with the controls (K). (L) SAOS2 cells were transiently co-transfected with a luciferase reporter construct containing either the HD (blue) or the PD (red) domain and either pJoP6 or pJoP6rec, which express Pax6. Luciferase activities are shown as relative values compared with the activity measured in lysates from cells co-transfected with the pJoP6 control plasmid. Co-transfection of pJoP6rec with either the HD- or the PD-containing reporter construct induced an increase in luciferase activity, indicating functionality of transgenic Pax6. Error bars indicate s.d.