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Methylation and luciferase reporter query


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#1 Prof Z

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Posted 03 October 2010 - 11:22 AM

Hi everyone, Was wondering if anyone had insights into the following...


1. Can normal pGL3 luciferase reporter plasmids (that have not been
SssI-methylase treated) also undergo methylation during transient
transfections?

2. I have transfected pGL3 under the human E-cadherin promoter into
Control and HPV-expressing cells. I notice a 50 % difference between
the two luciferase activities...would i be wrong in hypothesizing
methylation of the E-cadherin promoter is involved in the reduction? I am currently doing bisulfite sequencing on DNA from control and HPV expressing cells....fingers crossed!

I really look forward to hearing your thoughts on this....will be much
appreciated!

Kindest regards,

ProfZ

#2 methylnick

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Posted 03 October 2010 - 12:41 PM

ProfZ,

it is indeed possible that you could get invitro methylation of your transfected construct. And this is cell line specific.

Could it be that HPV is somehow modulating the E-cadherin promoter in an upstream manner? I presume HPV=Human Papliloma Virus?

Checking the methylation of these cells is the first point of call, and would be interested to see what you find, the other way is to in-vitro methylate your construct before transfection with SssI methylase.

nick

#3 Prof Z

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Posted 04 October 2010 - 12:13 AM

Hi Nick,

Yes, HPV is indeed the human papillomavirus. Will let you know in a week or two what happens with the bisulfite sequencing! :D

I am a bit unclear as to how in vitro methylating my construct will help address the question as to how HPV maybe regulating it? Wouldn't it just shut down promoter activity in control and HPV cells? (surely, i am missing something big here!)

Once again, thank you for your help :)

Z

#4 methylnick

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Posted 05 October 2010 - 05:05 PM

one would expect if you have already methylated your construct before you transfect you should see the same effect (reduction in luciferase activity) and if HPV is playing a role, potentially a synergistic effect of more silencing of your promoter and luciferase readout, instead of a 50% reduction, you would expect to get a 100%.

Maybe I have missed the big picture on my end about your expt~! :unsure:

#5 Prof Z

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Posted 19 October 2010 - 05:41 PM

Hi Methylnick,

Thank you for your reply :) I got back my sequencing results of my bisulfite product...but I am unsure how to proceed from now on. I put my product into pGEM-T-Easy vector from promega and sequnced it with Sp6 primer...(T7 primer sequencing terminated quite abruptly). Can you please let me know how to proceed from here? I notice people look at heights of peaks and ratios and so on and so forth?

Thank you for the trouble! Much appreciated!

Z




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