iPSCs
more convenient and less invasive to obtain than other cell types, such as fibroblasts. 33 Plasmid-based expression also eliminates the need for factors that may cause the expression of oncogenes, such as n- Myc. In addition, plasmids have a smaller genomic footprint than integrating viral vectors, which can be beneficial when studying gene function in iPSCs. 34 Plasmid-based expression to generate induced pluripotent stem cells (iPSCs) has been found to have several drawbacks. One major issue is that this method has lower efficiency rates for generating iPSCs, often several orders of magnitude lower than other methods. Additionally, the process involves the serial transfection of one or two plasmids that express the reprogramming factors of interest, which can be time-consuming and difficult to optimize. Furthermore, plasmids can persist in target cells and cause problems, as they may continue to make unwanted edits even after being diluted with each cell division. This persistence may also make it challenging to remove plasmids from cells entirely. Therefore, while plasmid-based expression has its advantages, it is essential to also consider its downsides carefully before deciding on a method for generating iPSCs. 35
The oocyte reprogramming method of iPSC generation
The process of generating stem cells in this method involves the introduction of specific transcription factors into a mammalian egg cell or oocyte. These transcription factors are Oct4, Sox2, Klf4, and c-Myc, and they play a crucial role in reprogramming the cell's genome to form stem cells. Unlike other methods of generating stem cells, this technique uses a different type of base cell, which is instrumental in the successful creation of these cells. The introduction of these transcription factors can be achieved through several techniques such as electroporation, microinjection, and gene transfer using adeno- associated virus vectors, among others. The successful reprogramming of the cell's genome is dependent on several factors, including the quality of the base cell, the efficiency of the technique used, and the experience of the researcher. Despite the complexity of this process, it holds great promise for the development of new treatments and therapies for a wide range of diseases and conditions. 36
Benefits and drawbacks
Oocyte reprogramming is a highly efficient and reliable method for generating iPSCs. This method has several advantages compared to other methods of iPSC generation. Firstly, oocyte reprogramming
33 Si- Tayeb, K., Noto, F.K., Šepac, A., Sedlić, F., Bosnjak, Z.J., Lough, J.W., & Duncan, S.A. (2010). Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors. BMC Developmental Biology, 10 , 81 - 81. 34 Capetian, P., Azmitia, L., Pauly, M.G., Krajka, V., Stengel, F., Bernhardi, E., Klett, M., Meier, B., Seibler, P., Stanslowsky, N., Moser, A., Knopp, A., Gillessen ‐ kaesbach, G., Nikkhah, G., Wegner, F., Döbrössy, M.D., & Klein, C. (2016). Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts. Frontiers in Cellular Neuroscience, 10 . 35 Si- Tayeb, K., Noto, F.K., Šepac, A., Sedlić, F., Bosnjak, Z.J., Lough, J.W., & Duncan, S.A. (2010). Gen eration of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors. BMC Developmental Biology, 10 , 81 - 81. 36 Guan, J., Wang, G., Wang, J., Zhang, Z., Fu, Y., Cheng, L., Meng, G., Lyu, Y., Zhu, J., Li, Y., Wang, Y., Liuyang, S., Liu, B., Yang, Z., He, H., Zhong, X., Chen, Q., Zhang, X., Sun, S., Lai, W., Shi, Y., Liu, L., Wang, L., Li, C., Lu, S., & Deng, H. (2022). Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature, 605 , 325 - 331.
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