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Title: Dissecting gene regulatory networks in vertebrate development using genomic and proteomic approaches
Keywords: Gene Regulatory Networks, Osteo-chondro specification, Vertebrate development, Cis-regulatory elements, Conserved Non-coding Elements, Epitope tagging
Issue Date: 31-Jul-2009
Citation: VISHNU RAMASUBRAMANIAN (2009-07-31). Dissecting gene regulatory networks in vertebrate development using genomic and proteomic approaches. ScholarBank@NUS Repository.
Abstract: The development of a multi-cellular organism from a single-celled fertilized egg is an autonomous process, requiring no instructions from the environment in which it develops. So the program specifying the instructions for the development of an organism lies hidden in the genome. In any cell, it is the specific combination of transcription factors present; in the context of its environment that defines the identity of the cell. It is these 2 components, the transcription factors and the cis-regulatory elements that read the regulatory state of a cell that form the Gene Regulatory Networks (GRNs) which control development. Studying gene regulatory networks involves the identification of the transcription factors expressed and the cis-regulatory elements that are active in a particular cell lineage. It also involves studying gene interactions at the transcriptional regulatory level and at protein interaction level. GRNs for certain lineage specification have been mapped in detail in invertebrate systems like sea urchin and in certain in vitro model systems for vertebrates. Studying GRNs in vertebrate development poses various challenges, arising from the complexity of the genome and the body plans of vertebrates. This necessitates the development of novel approaches to study GRNs in development. Developments in transgenic methods, genomic and proteomic technologies have opened new vistas for exploring gene regulatory networks in detail. Whole genome gene expression profiling using microarrays and mass spectrometry based methods for identification of protein-protein interaction and massively parallel sequencing methods for mapping transcription factor binding sites are some of the new developments that enable us to dissect gene regulatory networks. My projects involve developing methods and strategies to study GRNs in vertebrate development. One of the projects involves developing technology to isolate cells of a specific lineage from a mixture of other cells in the developing mouse embryo and study the gene regulatory pathway involved in the specification process. In a collaborative effort with in the lab, we have successfully generated Sox9+/+, Sox9+/- and Sox9 -/- chimeras expressing EGFP in Sox9 expressing cells in the developing mouse embryo. For studying the chondrogenic specification pathway, for which Sox9 is a master regulator, we have obtained whole genome gene expression data from sorted EGFP+ cells of all the three genotypes at E13.5 and E12.5 stages. Several differentially expressed genes between the three genotypes and the two time points have been identified. This includes well known targets of Sox9 and other known factors involved in osteo-chondro lineage development. Further studies are required to dissect out the GRN involved in this developmental pathway. My second project aims to develop and refine a method to identify long and short range cis-regulatory elements for developmental genes. These elements are often hidden in the vast deserts of non-coding DNA in vertebrate genomes. Computationally predicted conserved non-coding elements are assayed in vivo in developing zebrafish embryos for regulatory activity. A strong forebrain enhancer for the dlx5a/dlx6a bi-gene cluster in zebrafish has been identified. Enhancers driving the expression of this gene pair in other domains are yet to be identified. And finally, my other project involves developing a method for generating ES cell lines expressing epitope tagged transcription factors for mapping protein-protein interaction networks involved in pluripotency in mouse ES cells. Oct4-2xFlag-TEV-BAP expressing lines have been successfully generated. This can be used for TAP-MS analysis of the pluripotency network.
Appears in Collections:Master's Theses (Open)

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