Promoter Cloning for Luciferase Assay (View forum version)



matoge

Posted 17 November 2009 - 03:13 PM

Hi,

I want to investigate a putative TF for a promoter with the Luciferase Assay. I am about to clone the promoter into the Luciferase vector. The +1 site of that promoter is followed by a 5'-UTR. My question is now, whether I should include downstream sequences and if yes how much of it (50, 100bp)? Or is it better, that my sequence stops at -1 directly followed by the luciferase gene.

To reformulate the question:
Does the region around +1 have any important sequence information for the transcription machinery?
Could it matter if I have additional 50-100bp upstream in the the luciferase assay in terms of the transcription of the gene or the stability of the mRNA? Does anybody have experience with that?

Thanks,

Manuel

Sank

Posted 24 November 2009 - 02:03 PM

Hi Manuel
Generally people including exon 1 along with the 5' upstream region. If your gene has a TATA box, u may avoid the downstream region. For TATA-less promoters, u definitely have to include the downstream region.
Hope this helps.

Regards
Sankella

Hi,

I want to investigate a putative TF for a promoter with the Luciferase Assay. I am about to clone the promoter into the Luciferase vector. The +1 site of that promoter is followed by a 5'-UTR. My question is now, whether I should include downstream sequences and if yes how much of it (50, 100bp)? Or is it better, that my sequence stops at -1 directly followed by the luciferase gene.

To reformulate the question:
Does the region around +1 have any important sequence information for the transcription machinery?
Could it matter if I have additional 50-100bp upstream in the the luciferase assay in terms of the transcription of the gene or the stability of the mRNA? Does anybody have experience with that?

Thanks,

Manuel


StevieRay

Posted 24 November 2009 - 02:12 PM

Hi Manuel
Generally people including exon 1 along with the 5' upstream region. If your gene has a TATA box, u may avoid the downstream region. For TATA-less promoters, u definitely have to include the downstream region.
Hope this helps.

Regards
Sankella

Hi,

I want to investigate a putative TF for a promoter with the Luciferase Assay. I am about to clone the promoter into the Luciferase vector. The +1 site of that promoter is followed by a 5'-UTR. My question is now, whether I should include downstream sequences and if yes how much of it (50, 100bp)? Or is it better, that my sequence stops at -1 directly followed by the luciferase gene.

To reformulate the question:
Does the region around +1 have any important sequence information for the transcription machinery?
Could it matter if I have additional 50-100bp upstream in the the luciferase assay in terms of the transcription of the gene or the stability of the mRNA? Does anybody have experience with that?

Thanks,

Manuel

How long is your 5'-UTR? If its short (like mine was---about 50 bp), then make the ATG of the putative protein product match the ATG of luciferase, so as to include the 5' UTR.

Why do this? Becuase there are core promoter elements such as the "DPE" or "MTE" that lie downstream of the transcription start site(s), that are found in the 5'UTR region. I would do the same thing even if your promoter has a TATA box. There could still be important core promoter elements just downstream of the TSS.

I suggest reading review articles by Jim Kadonaga to learn about core promoter elements other than the TATA box, before you think about designing the luc reporter construct.

toprak

Posted 08 December 2009 - 08:27 AM

I cloned lots of luciferase reporter plasmids. I usually make more than one construct, for example with UTR, without UTR and also I take upstream regions of different sizes from a very short one to 6-7kb.