| dc.description | Stem-cells based regenerative therapy is one of promising alternative organ replacement therapy in the future. There are several approaches that can be applied on this technology
IPS-cells utilization also one of Stem-cells derived regenerative therapy technology which also support wider application in regenerative therapy and organ replacement.
In order to regenerate tooth using IPS-cells, several signaling cues are required for inducing proper tooth regeneration. Therefore, the understanding of molecular mechanism is inevitable.
SP6 is one of critical molecules for proper tooth development, truncated form of SP6 due to frameshift mutation exhibit specific phenotypical defect of Amelogenesis imperfecta, while SP6 transgenic rat showed yellowish tooth and accumulation of iron deposition in addition to the phenotype of delayed ameloblast maturation histopathologically. These evidences suggested the critical role of SP6 during tooth development.
My studies utilize G5 cells-dental epithelial derived cells, suggested the possible existence of dynamic and tight control of SP6 in the cellular context, since SP6 is very short-lived protein in G5 cells due to proteasomal degradation machinery. It also suggested the possibility of several co-regulators or binding-partner to exhibit the dynamic controlling mechanism of SP6.
Currently, there are limited information about SP6 binding partner or interacting molecules. Therefore, on this research I want to investigate the possibility for SP6 interacting molecules further.
On this study, I utilized SP6 stable transformant-cells which constitutively express SP6 and also derived from dental epithelial cells. After confirming and selecting the highest SP6 producer cells, I confirmed the expression of several candidate molecules for positive control and also prepared the nuclear extract fractionation.
In order to enrich the SP6 transcription expression, I planned to utilize nuclear extract sample. Surprisingly, I found interesting result related to SP6 localization under the cells-stress environment which maybe induced by proteasomal inhibitor. This finding also suggested some other possibility for SP6-down target genes regulation by SP6 in over-expression condition.
Furthermore, still the co-partner or SP6 possible interacting molecules is required to be investigated. Therefore, I did in vivo cross-linking procedure to fixate the complex molecules before performing immunoprecipitation. My initial cross-linking procedure showed some aggregated molecules complex was occurred within my sample compare to the non-crosslinked sample. Using this material, I further analyze by immunoprecipitation procedure to enrich the obtained crosslinked sample for targeting molecules complex.
Since our starting material are SP6 stable transformant cells, and this SP6 was already contain HA-tag. Then, we procced the pilot immunoprecipitation procedure using anti-HA high affinity antibody and confirm the result by different HA-probe antibody in addition to anti SP6 antibody for the identity. | en_US |
