Generation and characterization of rat and mouse monoclonal antibodies specific for MeCP2 and their use in X-inactivation studies

• Published in: PloS one 2011-11, Vol.6 (11), p.e26499-e26499
• Main Authors: Jost, K Laurence, Rottach, Andrea, Milden, Manuela, Bertulat, Bianca, Becker, Annette, Wolf, Patricia, Sandoval, Juan, Petazzi, Paolo, Huertas, Dori, Esteller, Manel, Kremmer, Elisabeth, Leonhardt, Heinrich, Cardoso, M Cristina
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Antibody Specificity - immunology; Antigens - immunology; Biology; Brain; Brain - cytology; Brain - metabolism; Chromatin; Chromatin Immunoprecipitation; Chromosomes; CpG islands; Cross Reactions - immunology; Deactivation; Deoxyribonucleic acid; DNA; DNA methylation; Epigenetics; Epitope Mapping; Female; Gene mapping; Genetic aspects; Heterochromatin; Heterozygote; Humans; Immunoblotting; Immunofluorescence; Immunology; Immunoprecipitation; Inactivation; Male; MeCP2 protein; Methyl-CpG binding protein; Methyl-CpG-Binding Protein 2 - immunology; Mice; Molecular Sequence Data; Monoclonal antibodies; Mutation; Nervous system diseases; Patients; Protein binding; Rabbits; Rats; Rett syndrome; Rodents; Sensitivity analysis; Species Specificity; X Chromosome - genetics; X Chromosome Inactivation - immunology; X chromosomes; X-chromosome inactivation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0026499

Methyl CpG binding protein 2 (MeCP2) binds DNA, and has a preference for methylated CpGs and, hence, in cells, it accumulates in heterochromatin. Even though it is expressed ubiquitously MeCP2 is particularly important during neuronal maturation. This is underscored by the fact that in Rett syndrome, a neurological disease, 80% of patients carry a mutation in the MECP2 gene. Since the MECP2 gene lies on the X chromosome and is subjected to X chromosome inactivation, affected patients are usually chimeric for wild type and mutant MeCP2. Here, we present the generation and characterization of the first rat monoclonal MeCP2 specific antibodies as well as mouse monoclonal antibodies and a rabbit polyclonal antibody. We demonstrate that our antibodies are suitable for immunoblotting, (chromatin) immunoprecipitation and immunofluorescence of endogenous and ectopically expressed MeCP2. Epitope mapping revealed that most of the MeCP2 monoclonal antibodies recognize the C-terminal domain and one the N-terminal domain of MeCP2. Using slot blot analysis, we determined a high sensitivity of all antibodies, detecting amounts as low as 1 ng of MeCP2 protein. Moreover, the antibodies recognize MeCP2 from different species, including human, mouse, rat and pig. Lastly, we have validated their use by analyzing and quantifying X chromosome inactivation skewing using brain tissue of MeCP2 heterozygous null female mice. The new MeCP2 specific monoclonal antibodies described here perform well in a large variety of immunological applications making them a very valuable set of tools for studies of MeCP2 pathophysiology in situ and in vitro.