Browsing by Subject "MEFs"
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Item Direct Reprogramming Of Fibroblasts Into Muscle Or Neural Lineages By Using Single Transcription Factor With Or Without Myod Transactivation Domain(2014-02) BELUR, NANDKISHORE RAGHAVThe generation of induced pluripotent stem cells (iPSCs) from somatic cells has opened new doors for regenerative medicine by overcoming the ethical concerns surrounding embryonic stem (ES) cell research. However, iPSC technology presents several safety concerns, such as the potential risk of tumor formation, that have caused apprehension for use of iPSCs in humans. One such approach is "direct reprogramming" which can bypass the iPSC or pluripotent stage and obtain tissue-specific cell types from somatic cells. In this study, we examined whether an important transcription factor involved in myogenesis (Pax3) or neurogenesis (NeuroD1) alone can directly reprogram the mouse embryonic fibroblasts (MEFs) into myogenic or neurogenic lineages, respectively. In addition, we created fusion transcription factors (Pax3 or NeuroD1) with the potent MyoD transactivation domain (MDA) that could facilitate radical acceleration of reprogramming into the desired cell type through chromatin modification compared to wild-type factors. Here, we showed that Pax3 can reprogram MEFs towards a myogenic lineage and that MDA-Pax3 further enhances this myogenic reprogramming event. In addition, ectopic expression of NeuroD1 and MDA-NeuroD1 is able to induce neurogenic genes in MEFs, suggesting the partial neurogenic conversion of MEFs. Furthermore, we also showed that the ectopic expression of NeuroD1 but not MDA-NeuroD1 in myoblasts could suppress myogenic differentiation. These data suggest that single gene transduction such as Pax3 or NeuroD1 will become a feasible therapeutic approach for neuro- and muscle degenerative diseases, respectively.Item Preparation of Induced Pluripotent Stem Cells from Fibroblasts under Circadian Rhythm(2020-12) Gupta, AbheepsaiPSCs are conventionally generated under constitutive promoters for a constant expression of pluripotency factors, yet the yield of reprogrammed cells is low. Since somatic cells are entrained in a circadian rhythm that regulates their metabolic and physiological functions, we hypothesize that the expression of the reprogramming factors should be done under circadian rhythm instead of a continuous expression. Doing this might hasten the process and improve the yield of iPSCs. To test this, I prepared plasmid constructs with the OSKM reprogramming cassette attached downstream of the circadian promoters Per2 or Bmal1. However, I could not obtain successful midipreps of these plasmids due to time constraints. Nevertheless, iPSCs were generated from fibroblasts with plasmids containing the OSKM genes under the constitutive LTR promoter. This independent experiment helped me become familiar with the process of virus generation and transduction and to determine the optimum seeding density to achieve iPSC formation from fibroblasts.