iPSCs
reprogramming factors are transmitted on a single plasmid less time is needed for the factors to enter the cell, thus increasing the efficiency of this method. 27
This method has several potential drawbacks. The use of polycistronic minicircle DNA vectors requires the expression of multiple genes, which can be challenging to control. Additionally, this method can lead to the formation of aberrant chromosomal structures and the generation of off-target effects. 28 Compared to other non-integrating reprogramming methods, the technology of using polycistronic minicircle DNA nonviral vectors is more complex meaning that it requires specific training to use. 29 Additionally, minicircles are removed from cells by dilution with each cell division, but it can still take several passages for the minicircle to be completely removed. Due to the minicircle vectors staying in the cells too long the use of this method can lead to the generation of incorrectly reprogrammed cells.
The plasmid-based expression method of iPSC generation
Plasmid-based expression is a method of expressing genes in cells using plasmids. These are small circular DNA molecules that can replicate independently of chromosomal DNA. 30 The plasmids are engineered to contain specific genes and elements that control gene expression. These plasmids are then introduced into cells, where they are used as templates for protein synthesis. Plasmid-based expression systems have been developed to use in a wide range of organisms, including bacteria, yeast, plants, and animals. These plasmids are used to generate the 4 key factors (Oct4, Sox2, Klf4, and c-Myc) inducing pluripotency in the target cells. 31
Benefits and drawbacks
The advantages of this method are that it is relatively simple to implement and does not require time- consuming production of virus. Plasmid-based expression is a non-integrating method, meaning it does not integrate into the genome, which reduces the risk of genetic errors. 32 Plasmid-based expression has several other benefits. It uses peripheral blood mononuclear cells (PBMCs) which are
27 ‘ Human Virology & Retrovirology. ’ - MedCrave online. MedCrave Publishing. Accessed March 1, 2023. https://medcraveonline.com/JHVRV/JHVRV-02-00044; Kenkel, Beth. ‘ Delivery Methods for Generating Ipscs. ’ Addgene blog. Accessed March 1, 2023. https://blog.addgene.org/delivery-methods-for-generating-ipscs. 28 Jia, F., Wilson, K.D., Sun, N., Gupta, D.M., Huang, M., Li, Z., Panetta, N.J., Chen, Z., Robbins, R.C., Kay, M.A., Longaker, M.T., & Wu, J.C. (2010). A nonviral minicircle vector for deriving human iPS cells. Nature Methods, 7 , 197-199. 29 Deng, X., Wang, H., Wang, T., Fang, X., Zou, L., Li, Z., & Liu, C. (2015). Non-Viral Methods For Generating Integration-Free, Induced Pluripotent Stem Cells. Current Stem Cell Research & Therapy, 10 , 153 - 158. 30 Chou, B., Mali, P., Huang, X., Ye, Z., Dowey, S.N., Resar, L., Zou, C., Zhang, Y.A., Tong, J., & Cheng, L. (2011). Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Research, 21 , 518 - 529. 31 Kriz, A., Schmid, K., Baumgartner, N. et al. A plasmid-based multigene expression system for mammalian cells. Nat Commun 1 , 120 (2010). https://doi.org/10.1038/ncomms1120 32 Chou, B., Mali, P., Huang, X., Ye, Z., Dowey, S.N., Resar, L., Zou, C., Zhang, Y.A., Tong, J., & Cheng, L. (2011). Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures. Cell Research, 21 , 518 - 529.
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