Research Awards
Paul Wade, Ph.D.
Emory University
"Methylated DNA binding proteins: Structure, function, and molecular interactions"
2-Year Award: $100,000
Research Sponsor for Both Years: The Massachusetts Rett Syndrome Association
Final Report (December 2002):
Specific Aim 1 proposed to create novel, improved reagents for the study of MeCP2 and other members of the MBD family. We have embarked on the production of monoclonal antibodies to full-length human MeCP2. Three mice were immunized and subsequently boosted three times. All three mice exhibited very high titer antibodies against MeCP2 as measured by ELISA. We performed two separate fusions to produce hybridoma lines. Following the first fusion, our screening and subcloning process resulted in the isolation of four clonal lines that produce antibodies against human MeCP2. Preliminary epitope mapping demonstrates that the epitopes for two of these antibodies are in the far carboxyl terminus of the protein ñ beyond amino acid 294. The epitopes for the remaining two antibodies lie within the first 168 amino acids of the protein. We thus have two C-terminal antibodies and two N-terminal ones. In addition, the second fusion yielded 3 additional hybridomas that are TRD specific.
Our evaluation of these antibodies determined that hybridoma supernatants were too dilute to be useful for immunofluorescence or immunoblotting. We plan to produce ascites in mice to obtain more concentrated antibodies.
Specific Aim 2 proposed to characterize the consequences of a subset of Rett Syndrome mutations in MeCP2 to its structure, stability and function.
We have generated 15 missense mutations in the MBD domain of MeCP2. These mutants can be expressed as full-length recombinant protein in bacteria, as MBD domain recombinant protein in bacteria, or in mammalian cells with a FLAG epitope tag. We are currently systematically determining DNA binding properties, structural consequences of mutation, stability, cellular localization, and association with Sin3A and HDAC1 for each of these mutants.
We have used the preliminary data assembled during the RSRF funding period into an RO1 proposal for NIH. This grant application was submitted in February, and received good scores just out of the fundable range. We have resubmitted a revised application which is currently pending. If this proposal is funded by NIH, we will continue with the studies initiated during our RSRF funding period.
Paul Wade, Ph.D.Emory University
"Methylated DNA binding proteins: Structure, function, and molecular interactions"
2-Year Award: $100,000
Research Sponsor for Both Years: The Massachusetts Rett Syndrome Association
Final Report (December 2002):
Specific Aim 1 proposed to create novel, improved reagents for the study of MeCP2 and other members of the MBD family. We have embarked on the production of monoclonal antibodies to full-length human MeCP2. Three mice were immunized and subsequently boosted three times. All three mice exhibited very high titer antibodies against MeCP2 as measured by ELISA. We performed two separate fusions to produce hybridoma lines. Following the first fusion, our screening and subcloning process resulted in the isolation of four clonal lines that produce antibodies against human MeCP2. Preliminary epitope mapping demonstrates that the epitopes for two of these antibodies are in the far carboxyl terminus of the protein ñ beyond amino acid 294. The epitopes for the remaining two antibodies lie within the first 168 amino acids of the protein. We thus have two C-terminal antibodies and two N-terminal ones. In addition, the second fusion yielded 3 additional hybridomas that are TRD specific.
Our evaluation of these antibodies determined that hybridoma supernatants were too dilute to be useful for immunofluorescence or immunoblotting. We plan to produce ascites in mice to obtain more concentrated antibodies.
Specific Aim 2 proposed to characterize the consequences of a subset of Rett Syndrome mutations in MeCP2 to its structure, stability and function.
We have generated 15 missense mutations in the MBD domain of MeCP2. These mutants can be expressed as full-length recombinant protein in bacteria, as MBD domain recombinant protein in bacteria, or in mammalian cells with a FLAG epitope tag. We are currently systematically determining DNA binding properties, structural consequences of mutation, stability, cellular localization, and association with Sin3A and HDAC1 for each of these mutants.
We have used the preliminary data assembled during the RSRF funding period into an RO1 proposal for NIH. This grant application was submitted in February, and received good scores just out of the fundable range. We have resubmitted a revised application which is currently pending. If this proposal is funded by NIH, we will continue with the studies initiated during our RSRF funding period.