Fig. 2

Generation and phenotypes of ERα S216A KI (Esr1^S216A) mice. a Map of strategy and Knock-in process. b Southern blot analyses were performed to identify the appropriately targeted allele. Genomic DNAs from mouse ear biopsies were digested with KpnI and BglI. KpnI-digestion generated 10.2- and 6.5-kb bands for knock-in (KI) and wild type (WT), respectively, detected by using the 5′-genomic probe. c Genotyping by PCR. Genomic DNA was isolated from mouse ear biopsies. PCR primers were used to amplify across the region in which the one remaining loxP site was inserted. This gave a band of 430 bp for a knock-in allele and 307 bp for a wild-type allele. Self-excision of the ACN cassette in the mutant allele was confirmed. d Expression of ERα mRNA. RNAs were prepared from WT and KI uteri and subjected to real time PCR analysis. These are averages from duplicates of two independent samples. e Conformation of ERα protein expression in WT and KI uteri. Whole extracts (15 μg proteins) prepared from WT and KI uteri were subjected to Western blots by an anti-ERα, an anti-P-Ser-216 (P-S216) or an anti-β-Actin antibody. f Sequence conformation of the mutation. cDNAs were synthesized from WT and KI uteri and sequenced. g ERα expression in ERα KI microglia. Accumulated microglia in the glia cells, which were prepared from brains of 2 days-old neonates of ERα KI males, were subjected to fluorescence staining with an anti-ERα (in green) antibody or double staining with an anti-Iba-1 (in red) antibody. DAPI stained nuclei in blue. h Obese phenotype of ERα S216A KI mice. Six-month-old mice were weighed; 5 males and 5 females of WT and 8 males and 4 females of KI mice. One Way ANOVA plus post hoc test with Tukey-Kramer’s multiple comparisons test (Version 5.0, Stat view-j) was used for statistical analysis. Values are presented as means ± S.E.. **, p < 0.01