Genetic reporter assays to study the regulation of interleukin-7 receptor alpha gene expression
Kurtuluş, Sema (2005) Genetic reporter assays to study the regulation of interleukin-7 receptor alpha gene expression. [Thesis]
Interleukin-7 (IL-7) signaling through IL-7 receptor controls survival and proliliferation of B- and T- lymphocytes at early stages of development. IL-7 is also a critical survival factor for naive T-lymphocytes and memory T-cells circulating in the peripheral organs. Throughout T-cell development, IL-7Ralpha expression on distinct T-cell populations is strictly regulated. However, the mechanisms that regulate differential expression of the IL-7Ralpha chain on T- lymphocytes are poorly defined. Here, we used bioinformatic tools to determine possible cis-acting regulatory elements at the downstream of mouse IL-7Ralpha gene and we tried to analyze the transcriptions! activities of these possible cis-regulatory elements in a mouse T lymphoma cell line by means of genetic reporter systems We took advantage of the luciferase assay system for functional analysis of noncoding sequences at the downstream of mouse IL-7Ralpha gene. We modified luciferase reporter vector by inserting weak promoter into the ups team of luciferase cDNA and cloned the mouse noncoding sequences into this modified vector. We electroporated mouse T lymphoma cells with luciferase reporter vectors to evaluate the effects of these noncoding regions on the transcription of luciferase cDNA. Although the luciferase activities in T lymphoma cell lysates were low due to low sensitivity of the detection system used, we observed that efficient transfections with luciferase reporter vectors resulted in high luciferase activities in a different cell line. We concluded that different reporter systems or different cell lines were required to determine possible transcriptional activities of the cloned mouse noncoding sequences.
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