Figure 1.

A SGIRT mouse model for conditional inactivation and restoration of Mecp2. A, ES targeting strategy. Exon 3 of Mecp2 encoding part of the methyl-CpG-binding domain was flanked by four pairs of recombinase recognition sites: lox2272, LoxN, Frt, and F5. One round of Cre- or Flp-mediated recombination inverts this exon (Mecp2^IVC or Mecp2^IVF), while two rounds of recombination restore its orientation (Mecp2^R). B, Southern blot confirmation of correctly targeted ES clone, which was used to generate Mecp2^CF/Y mice. A, Short black bar represents Southern probe. C, Genomic PCR confirmation of successful recombinations and changes of exon 3 orientation in different alleles. The positions of three primer pairs are shown in A. D, RT-PCR with primers encompassing exon 3 confirmed the exclusion of exon 3 in mRNA extracted from Mecp2^IVC/Y (shorter band) and the inclusion of exon 3 in mRNAs extracted from Mecp2^WT/Y, Mecp2^CF/Y, and Mecp2^R/Y brains (longer bands). E, Western blot confirmation of normal MeCP2 protein expression in Mecp2^CF/Y, loss of its expression in Mecp2^IVC/Y, and restoration of expression in Mecp2^R/Y. F, Indistinguishable body and brain weights between Mecp2^WT/Y, Mecp2^CF/Y, and Mecp2^R. n = 7. G, Representative photographs of brains taken from 8-week-old adult mice of the four genotypes showing comparable sizes of Mecp2^WT/Y, Mecp2^CF/Y, and Mecp2^R/Y, and reduced size in Mecp2^IVC/Y. The weights of these brains are (left to right) as follows: 413, 423, 351, and 417 mg. Scale bar, 500 μm. Data are mean ± SEM. Dots, squares, and triangles represent data from individual mice. n.s., Not significant at p > 0.05. For more statistical details, see Table 2.