CCNA2 Deletion Decreases Induced Neurons through 3D Nanochannel Electroporation
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Date
2015-03-25
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Abstract
Cell reprogramming is a switch in gene expression of a cell into an entirely different specialized cell type. Reprogramming can be induced artificially using exogenous transcription factors. One of the traditional methods to cause cell reprogramming is transfection by bulk electroporation (BEP), which introduces plasmid DNA into cells. However, BEP has many drawbacks, including low transfection efficiency, irreversibly damaging a large percentage of cells, and only transfecting a random assortment of cells. A novel technology, 3-dimensional nanochannel electroporation (NEP), allows for single cell transfection by applying a focused electric field through a nanochannel, precisely delivering DNA into a cell. This research project focuses on the use of 3D NEP for artificial cell reprogramming. Overexpressing transcription factors Brn2, Ascl1, and Myt1l (BAM) induce mouse embryonic fibroblasts (MEFs) into neurons, shown by the presence of TUJ1, a neuron-specific protein. Using BAM-mediated nuclear reprogramming, the role of type-A cyclins was investigated further. Cyclins are proteins that regulate the cell cycle by activating cyclin-dependent kinases. Cyclin A2, from the gene CCNA2, has been shown to be necessary to begin DNA replication, particularly the S-phase. Deletion of the CCNA2 gene locus decreases nuclear reprogramming, demonstrated by a lack of TUJ1-positive cells. Therefore, we concluded that CCNA2 is necessary for BAM-mediated nuclear reprogramming. Our results suggest that cells undergo S-phase and likely divide during reprogramming. These innovative results allow greater understanding regarding the cell reprogramming mechanisms, which are not well comprehended. The use of NEP for cell reprogramming is not only limited to DNA transfection. NEP may also be utilized with any charged molecule, such as drugs and RNA, underscoring its potential clinical applications in regenerative medicine, a rapidly advancing field.
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Health Professions - Laboratory/Cellular (The Ohio State University Denman Undergraduate Research Forum)
Keywords
cell reprogramming, regenerative medicine, cyclin A2, transfection