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Fig. 8. Conditional transgenic activation of Pax6-5a via the
JoP6-5a mouse line. (A,A') GFP fluorescence
on sections of E14.5 cortex of JoP6-5a (A) is partially lost in
JoP6-5a;Emx1IREScre (A'). (B) Two
products generated by genomic PCR with the primers JoP6F and JoP65aR (1955 bp
and 394 bp) confirm successful recombination in the
JoP6-5a;Emx1IREScre cortex, whereas the
JoP6 cortex and the pJoP6-5a plasmid show no recombination.
(C) Whole-mount ß-gal staining of E15.5 JoP6-5a and
JoP6-5a;Emx1IREScre brains shows strong
activation of the lacZ reporter in the
JoP6-5a;Emx1IREScre telencephalon and olfactory
bulb. (D,D' with inset) Immunostaining for Pax6 on E14.5
JoP6-5a and JoP6-5a;Emx1IREScre cortices
shows ectopic antibody staining indicating Cre-induced activation of
Pax6-5a in the JoP6-5a;Emx1IREScre CP.
(E) SAOS2 cells were transiently co-transfected with a luciferase
reporter construct containing either the HD (blue) or the PD (red) domain and
either pJoP65a or pJoP65arec, which express
Pax6-5a. Luciferase activities were normalized to co-transfections
with the pJoP65a 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 Pax6-5a. (F) q-PCRs with RNA isolated from E12.5 cortex of
JoP6-5a control and JoP6;Emx1IREScre
cortex, normalized to 18S RNA, indicate only a slight enhancement of the
Pax6-5a expression level in the
JoP6-5a;Emx1IREScre telencephalon and olfactory
bulb. Error bars indicate s.d.. (G,G') TUNEL assay on
E11.5 brain sections of both genotypes reveals no significant difference in
apoptosis (arrowheads point to endogenous apoptosis).
(H,H') BrdU immunohistochemistry after a 30-minute BrdU
pulse on cross-sections of E11.0 JoP6-5a and
JoP6-5a;Emx1IREScre cortices reveals a
significant inhibition of the proliferation rate in the
JoP6-5a;Emx1IREScre DP.
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