For example, to study the impact of promoter mutations on gene expression
First you have to isolate the promoter by PCR amplification.
In these amplifications you can generate in paralel, any promoters with variants or mutations and then cloned them into any expression vector containing no promoter (usually with the firefly luciferase gene).
After that, you have to transfect cells with these vectors and measure the amount of mRNA expressed via qPCR of luciferase gene or the luciferase activity using any kit of luminescence.
There are many variations to perform the analysis of promoters, maybe you should be more specific. Regards.
First you have to isolate the promoter by PCR amplification.
In these amplifications you can generate in paralel, any promoters with variants or mutations and then cloned them into any expression vector containing no promoter (usually with the firefly luciferase gene).
After that, you have to transfect cells with these vectors and measure the amount of mRNA expressed via qPCR of luciferase gene or the luciferase activity using any kit of luminescence.
There are many variations to perform the analysis of promoters, maybe you should be more specific. Regards.
Gracias Edgardo, era lo que me imaginaba. He visto que mi pregunta se ha quedado cortada.¿qué vector has usado en concreto?
Saludos
Aca usamos el sistema de reporteros pGL3 de Promega. Saludos.
I have found the following tools but I haven't used them:
Promoter predictions
PromoterInspector http://www.genomatix.de/online_help/help_gems/
PromoterInspector_help.html
Promoter prediction
FirstEF http://rulai.cshl.edu/tools/FirstEF Promoter prediction
DBTSS (Database of Transcriptional Start Sites)
http://dbtss.hgc.jp/index.html Transcriptional start site
TF-binding profile database
TRANSFAC® http://www.gene-regulation.com/pub/databases.html#
transfac
TF-binding site (matrix), TFs
TF-binding site prediction
TESS (Transcription Element Search
System)
http://www.cbil.upenn.edu/cgi-bin/tess/tess TF-binding site prediction
Match™ http://www.gene-regulation.com/cgi-bin/pub/programs/
match/bin/match.cgi
TF-binding site prediction
Regards
A standardized step-by-step promoter analysis could go something like this:
Determine the transcriptional start-site using 5'-RACE.
Evaluate evolutionary conservation to determine the extent of the promoter. In addition highly conserved clusters may reveal key regulatory elements, which can be evaluated in subsequent reporter analysis.
Reporter analysis of various promoter regions (deletion analysis) either transient or stable. Transient analysis is the more common approach, but there are several examples of non-integrated promoters (on plasmids) behave different than promoters integrated in the genome. Subsequently, the deletion analysis can be supplemented with a more specific analysis using point-mutated constructs. The target-sites can be chosen either based on evolutionary conservation or on candidate approach (e.g. matching the consensus of known transcription factors).
The promoter analysis of the generated constructs should be followed up by an evaluation of the promoter in its natural contest. This is most easily done by chromatin immuno-precipitation probing for specific histone marks and/or specific transcription factors. Preferably this would be done in two different contexts; one in which the promoter is active, and one in which it is silenced.
Finally, if you want to go all the way you will generate a transgenic mouse in which the expression of a reporter gene is driven by the DNA fragment that you have identified. The expression pattern of your reporter should then be compared with the endogenous expression pattern of your gene of interest, and only if it recapitulates the expression pattern 100% you can say the you have in your fragment all regulatory elements. In most cases your reporter will be expressed in only a subset of the tissues in which the gene is naturally expressed in which case you have identified the region responsible for expression in these specific tissues.
This latter step is commonly used in fly genetics, but much less common in mouse genetics (due to the time constraint.
I hope this was useful, best of luck
Strategy for promoter analysis ：1.Isolate putative promoter from genomic clone
2.Sequence putative clone 3.Determine transcription start site 4.Establish functional assays 5.Identify cis-acting DNA elements and trans-acting factors 6.Determine relevance of these factors 7.Establish regulatory model of the promoter in vivo and in vitro(According to the idea of SIBCB_Chen Wang)
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