Figure 7.

A humanized CTD2 allele expresses low levels of truncated MeCP2. (a) The amino acid sequences of the extreme C-termini of CTD1, CTD2 and CTD2hu mouse alleles and their corresponding human RTT alleles. Missense/nonsense changes caused by the deletion-frameshift are in bold type. Residues where the mouse allele differs from human are shown in red. Changes between CTD2 and CTD2hu are underlined. (b) Western blot showing levels of MeCP2 protein present in neurons on day 15 of differentiation (7 days after plating). Lanes A, B (and C) for each genotype are derived from independent mouse ES cell clones. An antibody against the N-terminus of MeCP2 detects both full-length and truncated (Δ) protein. (c) Quantification of MeCP2 levels in in vitro differentiated neurons. Two or three independent clones were differentiated for each genotype. MeCP2 level (MeCP2 signal/H3 signal) is normalized to the mean WT+loxP value. Data shown are mean (black line) and individual values for three (WT+loxP, CTD2hu) or two (CTD1, CTD2) independent differentiations. Comparison to WT+loxP (two-tailed unpaired t-test with Welch’s correction for unequal variances): CTD1 P=0.0108 (*), CTD2 P=0.5450 (ns) and CTD2hu P=0.0111 (*). (d) Western blot showing levels of MeCP2 protein present in LUHMES-derived human neurons on day 9 after precursor plating. Differentiations of one MECP2-null clone, two WT pools, two unmodified WT clones and three CTD2 LUHMES clones are shown. An antibody against the N-terminus of MeCP2 detects both full-length and truncated (Δ) protein. (e) Quantification of MeCP2 levels in LUHMES-derived neurons. Three independent differentiations were performed for each of the clones shown in (d). MeCP2 level (MeCP2 signal/H3 signal) is normalized to the WT pool value for each differentiation. Data shown are mean (black line) and individual values for three independent differentiations. Comparison to WT A (two-tailed unpaired t-test): WT B P=0.0798 (ns), CTD2 A P=0.0015 (***), CTD2 B P=0.0039 (***), CTD2 C P=0.0027 (***).