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Brumi Brumi
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10 years ago
Can somebody explain to me the difference between transkriptional and translational fusion by reporter gene assay ?
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Staff Member
Educator
10 years ago
Transcriptional fusion is a gene construct that investigates transcription activity of a gene of interest. It can be carried on a plasmid vector or integrated into genome. Transcription fusions are made of promoterless reporter genes eg. lacZ or Alkaline phosphatase etc. fused to the sequence surrounding the promoter your gene of interest plus its ribosomal binding sequence. The logic then is that you can assay for expression of your reporter gene, which would be equivalent to expression of your gene of interest. You can avoid all this pain by simply doing quantitative real time PCR. This however does require careful manipulation of RNA, which is not that easy.
The translational fusion is a modification of the transcriptional construct. The translational fusion bears the promoter of your gene and other sequence surrounding it (C-terminal) as well as the N-terminal sequence of your gene. The reporter gene is then inserted between these two terminals and in-frame such that you have one long protein product. This construct supposedly be expressed differently if posttranscriptional modification occurs but I don't buy it too much. You are still assaying, in part, for some transcription with this construct however way you look at it. I think that the difference between transcriptional and translational fusion expression is a more accurate account of what is happening but your are still assuming that your construct has totally no effect on the system.
Mastering in Nutritional Biology
Tralalalala Slight Smile
wrote...
10 years ago
With a transcriptional fusion you tag a reporter after a promoter of interest, thus you can measure the transcriptional activity of the promoter. With a translational fusion you tag your protein of interest, which means you can analyze its localization within a cell and also learn more about its actual expression levels (since protein levels are regulated on many different levels, not only promoter activity).
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