Figure 1.

Inducible expression of dominant-negative RhoA (N19–RhoA) at the Mapt locus. A–C, Schematic indicating targeting and expression schemes: loxP, loxP sites; Tx Stop, transcriptional stop sequence; N19RhoA, HA-tagged N19–RhoA cDNA; pA, Pgk1 polyadenylation sequence; neo, G418-resistance cassette. Scheme is not to scale. A, Scheme indicating Cre-mediated induction of N19–RhoA expression. For details, see Results. B, Map of endogenous Mapt allele (top) and the targeting construct (bottom) that is inserted into exon 1 during homologous recombination. Endogenous Mapt genomic sequences are revealed by BamHI or KpnI digestion, followed by Southern blotting with 5′ or 3′external probes, respectively. C, Map of targeted Mapt allele, before (top) and after (bottom) Cre-mediated recombination. Genomic alterations caused by the knock-in construct are detected by Southern blotting with external 5′ or 3′ probes after BamHI or KpnI digestion, respectively. Cre-mediated recombination eliminates the floxed transcriptional stop sequence, as revealed by KpnI digestion and Southern blotting with a RhoA probe. D, E, Southern blot of ES cell clones using the 5′ (D) and 3′ (E) external probes. Targeted clones (t) show a wild-type band and a band corresponding to the targeted allele at 2.9 kb (D) and 8.1 kb (E). DNA markers are indicated (right) with size in kilobase pairs.