Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells

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Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells

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Title: Characterization and Development of Strategies for Altering Protein Expression in JSL1 Cells
Author: Senitko, Annette Nelson
Abstract: Alternative splicing is a common mechanism for regulating gene expression in eukaryotic cells. This process of differentially including or excluding variable exons provides a means for increasing proteome complexity. Alternative gene splicing occurs in a cell specific manner and may be influenced by changes in the extracellular environment. Despite the importance of this method for regulating gene expression, little is known about the factors involved in regulating its function. The T cell tyrosine phosphatase CD45 provides a valuable model for investigating the factors involved in regulating alternative splicing. The CD45 gene contains three variable exons whose splicing is regulated in response to T cell activation. Studies of this gene have revealed the presence of an exonic silencer sequence within variable exon 4 that is capable of influencing exon skipping under both resting and stimulated conditions. Biochemical assays have shown that the regulatory protein hnRNP L binds to this silencer sequence and results in basal exon repression during resting conditions and undergoes modifications which further influence exon skipping upon stimulation. Furthermore, in vitro assays indicate that upon stimulation, an additional regulatory protein, PSF, binds to the regulatory complex associated with the silencer sequence. Although these studies have provided novel information regarding the regulation of splicing, biochemical assays are unable to fully mimic the signaling pathways inside a cell, thus creating a need for a cell culture system. A Jurkat derived cell line, JSL1 cells, has been identified as being able to recapitulate the signal induced alternative splicing of the CD45 gene as seen in primary human T cells. This cell line presents a cell based system for evaluating the factors involved in splicing. However, in order to conduct in vivo experiments one must be able to modify protein expression. JSL1 cells present limitations due to difficulties in being able to alter protein expression. A strong promoter, EF1-alpha , has been employed to drive the expression of candidate proteins in JSL1 cells. Transient transfections and stable cell lines expressing cDNAs driven by this promoter have shown little if any overexpression of candidate proteins normally expressed at high levels within the cell; however, significant overexpression has been achieved with the transfection of at least one protein that exists at a lower concentration. Initial experiments indicate that stably expressed flag-tagged proteins, driven by the EF1-alpha promoter, may be easily purified from JSL1 cells during resting and stimulated conditions and analyzed. Such data suggests that this promoter may afford more flexibility in altering and analyzing protein expression in JSL1 cells, thereby facilitating the investigation of signaling pathways involved in regulating alternative splicing. Furthermore, strategies for regulating protein expression, through the use of a Tet-suppressor system, are in initial stages of being developed and hold the potential for providing an additional tool for evaluating the factors involved in regulating alternative splicing.
URI: http://hdl.handle.net/2152.5/241
Date: 2007-08-08

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