The range of IRES activity can be widened by mutating its sequence. ![]() Although expression can be altered by using different naturally available IRES, the range of expression levels obtained is narrow due to the limited range of IRES elements. More importantly, as genes are translated independently, the relative expression of different genes can be adjusted by varying the strength of the IRES applied to each gene. In contrast to the 2A element, products generated using IRES do not form any undesirable modified or fusion proteins. It has already been shown that IRES allows strict control of the relative gene expression in both transient and stable transfections. When IRES elements are included between multiple open reading frames (ORFs), the first ORF is translated by the canonical cap-dependent mechanism while the rest are translated through a cap-independent mechanism. Moreover, incomplete cleavage of 2A peptides often results in attachment of additional unwanted amino acids residues to proteins and formation of fusion proteins,. This method does not allow modulation of the expression ratio between the proteins of interest. 2A linked genes are expressed in one single open reading frame (ORF) and “self-cleavage” occurs co-translationally to give equal amounts of co-expressed proteins. This method requires elimination of cryptic splicing sites in protein coding sequences to prevent incorrect RNA splicing.Ĭo-expression of multiple genes in one mRNA for strict control of relative gene expression can also be achieved by using either 2A elements or internal ribosome entry site (IRES). The use of splicing signals allows stricter control of relative gene expression as all genes are expressed in one transcript. The degree to which gene expression is suppressed depends on the integration site in the genome. However, the expression ratio still varies between cells in a stably transfected cell pool, , as the arrangement of multiple promoters in close proximity causes transcriptional interference, where the active expression of one gene suppresses expression of the other genes. Using a single vector with multiple promoters or polyadenylation signals ensures the introduction of several genes into each cell at identical amounts and provides accurate control of gene expression in transient transfections. Co-transfection is an inaccurate approach as the relative amount of different genes incorporated varies from cell-to-cell due to variations in transfection efficiency. Three common strategies for controlling multiple gene expression in mammalian cells are (i) co-transfection of multiple vectors at different relative amounts, ,, (ii) single vector containing multiple promoters or polyadenylation signals with different strengths, ,, and (iii) insertion of splicing signals with varied splicing efficiencies between genes. ![]() Simultaneous expression of multiple genes in mammalian cells at finely controlled amounts or ratios is required for applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells,. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: This work was supported by the Biomedical Research Council/Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. Received: SeptemAccepted: OctoPublished: December 9, 2013Ĭopyright: © 2013 Koh et al. Jude Children's Hospital, United States of America (2013) An Internal Ribosome Entry Site (IRES) Mutant Library for Tuning Expression Level of Multiple Genes in Mammalian Cells. Citation: Koh EYC, Ho SCL, Mariati, Song Z, Bi X, Bardor M, et al.
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