Expression vectors based on modified ribosomal protein promoters and uses thereof in post-transcriptional assessment

摘要

The present invention relates to expression vector comprising (a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter, (b) an operably linked reporter or gene sequence, and (c) a 3′ untranslated region (3′ UTR), which are suitable means for an selective assessment of post-transcriptional regulation, post-transcriptional control elements and factors as well as for identifying compounds that effect post-transcription. The present invention furthermore relates to arrays, expression vector libraries and cell lines containing the expression vector(s). The present invention furthermore relates to a method and kit for identifying compounds that affect post-transcriptional regulation of reporter(s) or gene(s), that utilize the expression vector(s).

主权项

1. An in vitro method for assessing a post-transcriptional effect in a cell wherein said method comprises:
(i) providing an expression vector comprising:
(a) a promoter region comprising a non-inducible constitutively active ribosomal protein gene promoter and at least one sp1 site-containing sequence, wherein the ribosomal protein gene promoter comprises the promoter of ribosomal protein S23 (RPS23) gene or the promoter of ribosomal protein S30 (RPS30) gene, or a fragment of said RPS23 promoter or said RPS30 promoter wherein said fragment has at least 50 nucleotides;(b) a reporter gene or heterologous gene; and(c) a 3′ untranslated region (3′ UTR),wherein said reporter gene or heterologous gene is operably linked to said promoter region and said 3′ UTR; (ii) introducing the expression vector into a cell; and (iii) assaying reporter gene or heterologous gene expression in the transfected cell of step (ii) wherein expression of the reporter gene or heterologous gene is responsive to a post-transcriptional effect, and wherein the expression of said reporter gene or heterologous gene is independent of transcriptional induction by TNF-α or okadaic acid.