7 replies to this topic

[liweixie]

I am study in transcriptional regulation.
Before we cloned gene promoter into reporter vector and transfected the reporter vector into rat intestinal cell plus another HIF expression vector to express Hypoxia inducible factor which upregulate this gene expression. Result shows 4-6 time induction compared with the cells without the HIF vector co-transfection.
Now, we want tot see whether the endogenous gene has similar level induction as the promoter luciferase result.
We transfected HIF expression vector to the cell and then isolated the total RNA, synthesize the first strand cDNA with bio-rad kit and run qRT-PRC with the primer used for a long time for this gene. Result shows no induction.
I do not know what is wrong with the qRT-PCR. Both methods used to measure the transcription level, how ever show different result.
Do you have experience like this? We doubted that we missed something however have not identified what we missed.

[pcrman]

Hello,

It doesn't surprise me. The cloned promoter is very different from the endogenous promoter with regards to its promoter environment. First you are using plasmid to introduce the promoter to the cells which is not integrated into the genome, 2nd, the cloned promoter is short and lacks upstream regulatory sequences such ans silencers and enhancers. So reporter assay is a very artificial thing.

[arnabde2000]

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

[Dr Teeth]

arnabde2000, on Sep 28 2009, 02:25 AM, said:

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

In addition to needing other promoter elements (e.g. enhancer regions, etc.) as described by others above, the native promoter is also subject to higher order chromatin structure and promoter elements may be inaccessible to transcriptional machinery as a result.  This is why luciferase constructs of promoter regions are only useful as a crude tool and must be backed up with endogenous data.

[Dr Teeth]

Dr Teeth, on Sep 28 2009, 08:00 AM, said:

arnabde2000, on Sep 28 2009, 02:25 AM, said:

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

In addition to needing other promoter elements (e.g. enhancer regions, etc.) as described by others above, the native promoter is also subject to higher order chromatin structure and promoter elements may be inaccessible to transcriptional machinery as a result.  This is why luciferase constructs of promoter regions are only useful as a crude tool and must be backed up with endogenous data.

And just to be sure, you are subjecting the cells to hypoxia (physiological or chemically induced) before checking for induction, yes?

[liweixie]

Dr Teeth, on Sep 28 2009, 04:01 AM, said:

Dr Teeth, on Sep 28 2009, 08:00 AM, said:

arnabde2000, on Sep 28 2009, 02:25 AM, said:

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

In addition to needing other promoter elements (e.g. enhancer regions, etc.) as described by others above, the native promoter is also subject to higher order chromatin structure and promoter elements may be inaccessible to transcriptional machinery as a result.  This is why luciferase constructs of promoter regions are only useful as a crude tool and must be backed up with endogenous data.

And just to be sure, you are subjecting the cells to hypoxia (physiological or chemically induced) before checking for induction, yes?

Yes We tried different way to subject cell to hypoxia condition( chemical induce or everexpression) We can detect the Hif mRNA induction.
Here is some evidence that our animal model under iron deprevation condition, the cells were highly hypoxia. So, from animal model, most of the hypoxia target genes were regulated. However, back to cell, qRT-PCR does not show similar induction as animal model.

[Dr Teeth]

liweixie, on Oct 2 2009, 11:20 AM, said:

Dr Teeth, on Sep 28 2009, 04:01 AM, said:

Dr Teeth, on Sep 28 2009, 08:00 AM, said:

arnabde2000, on Sep 28 2009, 02:25 AM, said:

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

In addition to needing other promoter elements (e.g. enhancer regions, etc.) as described by others above, the native promoter is also subject to higher order chromatin structure and promoter elements may be inaccessible to transcriptional machinery as a result.  This is why luciferase constructs of promoter regions are only useful as a crude tool and must be backed up with endogenous data.

And just to be sure, you are subjecting the cells to hypoxia (physiological or chemically induced) before checking for induction, yes?

Yes We tried different way to subject cell to hypoxia condition( chemical induce or everexpression) We can detect the Hif mRNA induction.
Here is some evidence that our animal model under iron deprevation condition, the cells were highly hypoxia. So, from animal model, most of the hypoxia target genes were regulated. However, back to cell, qRT-PCR does not show similar induction as animal model.

Have you looked at endogenous gene induction in cell culture and are these working?  
Also of concern, you said "We can detect the Hif mRNA induction," but do you have a reference for this?  HIF mRNA is not induced by hypoxia, instead, the HIF protein is constitutively produced but under normoxia is constitutively targeted for degradation via the 26S proteasome via prolyl hydroxylation and VHL, meaning that mRNA levels are normal, but the protein exists at very low levels.  Under hypoxic conditions, the HIF protein is no longer targeted for degradation and thus accumulates quickly without the need for new transcription/translation events, allowing for the necessary rapid response.  Thus, this change in protein expression occurs without a concomitant change in mRNA levels.  In general, hypoxia leads to a global decrease in mRNA levels as part of the stress response with induction of those genes needed to "save" the tissue from hypoxia--VEGF, EPO, GLUT-1 etc.

[liweixie]

Dr Teeth, on Oct 5 2009, 04:49 AM, said:

liweixie, on Oct 2 2009, 11:20 AM, said:

Dr Teeth, on Sep 28 2009, 04:01 AM, said:

Dr Teeth, on Sep 28 2009, 08:00 AM, said:

arnabde2000, on Sep 28 2009, 02:25 AM, said:

Is it possible that your gene is under the expression of other additional elements besides the promoter? In the luciferase assay, the luciferase gene is only under the influence of the promoter.

In addition to needing other promoter elements (e.g. enhancer regions, etc.) as described by others above, the native promoter is also subject to higher order chromatin structure and promoter elements may be inaccessible to transcriptional machinery as a result.  This is why luciferase constructs of promoter regions are only useful as a crude tool and must be backed up with endogenous data.

And just to be sure, you are subjecting the cells to hypoxia (physiological or chemically induced) before checking for induction, yes?

Yes We tried different way to subject cell to hypoxia condition( chemical induce or everexpression) We can detect the Hif mRNA induction.
Here is some evidence that our animal model under iron deprevation condition, the cells were highly hypoxia. So, from animal model, most of the hypoxia target genes were regulated. However, back to cell, qRT-PCR does not show similar induction as animal model.

Have you looked at endogenous gene induction in cell culture and are these working?  
Also of concern, you said "We can detect the Hif mRNA induction," but do you have a reference for this?  HIF mRNA is not induced by hypoxia, instead, the HIF protein is constitutively produced but under normoxia is constitutively targeted for degradation via the 26S proteasome via prolyl hydroxylation and VHL, meaning that mRNA levels are normal, but the protein exists at very low levels.  Under hypoxic conditions, the HIF protein is no longer targeted for degradation and thus accumulates quickly without the need for new transcription/translation events, allowing for the necessary rapid response.  Thus, this change in protein expression occurs without a concomitant change in mRNA levels.  In general, hypoxia leads to a global decrease in mRNA levels as part of the stress response with induction of those genes needed to "save" the tissue from hypoxia--VEGF, EPO, GLUT-1 etc.

We use qRT-PCR to test the endogenous gene expression. In short, we transfected cells with the HIF-1&2 alpha construct. After 36 hours, RNA was isolated and RT-PCR. Endogenous  gene expression was tested by qRT-PCR. qRT-PCR primer is the one we used before, and it works fine for this gene.
We supposed that the gene was HIF target gene. However, qRT-PCR result show no induction.
The other question is about the HIF mRNA level in cell. Usually, the HIF level is low in cell. But we overexpressed the HIF cDNA in pcDNA3.0 in cell. The overexpressed HIF supposed to increase the HIF target gene expression. Resent published two paper, one on cell and the other on biomedical, talked about one of the HIF targeted gene, DMT1. They co-transfected HIF construct( we get from them) with the DMT1 promoter construct to the cells and let it overexpressed. Their result shows that there is no induction for HIF 1 and around 10 times induction for HIF 2. Anyway, they based on the HIF binding site on gene promoter region.
For HIF protein degradation in cell, the overexpressed HIF, its degradation rate is much lower than its expression rate. So, the alpha subunit reacts with the beta subunit and the dimer binds to the promoter region of the targeted gene to increase or decrease the expression. Besides our gene, we also test the VEGF as positive control, VEGF does not show any induction.