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Cloned but not expressing!


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8 replies to this topic

#1 mansi368

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Posted 11 February 2013 - 08:17 AM

The pEF6V5 his TOPO expression vector was used to clone a gene. We were able to get the sequence but it isnt showing any expression. The construct has EF1a promoter. it is constitutive. can u please suggest why? and an alternative solution to get it expressed.

#2 GNANA

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Posted 11 February 2013 - 09:27 AM

Did you tried transfecting in different cell lines and concluded it failed to express? because i have experienced some contructs express in 293 but not in others...this might be due to weaker kozak, so did u add kozak?? if everything is fine, sequence and check whether the insert is in frame.

Gnana...
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#3 jerryshelly1

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Posted 11 February 2013 - 05:00 PM

You have the correct sequence and everything is still in the correct frame? Is your cell line transient or stable? Try what GNANA said and transiently transfect an HEK or HeLa cell. They usually show expression of plasmids that would otherwise not show expression in your current cell line. What are you doing to determine the expression? Have your put this particular plasmid into your current cell line and saw expression with a different gene?

Good luck

#4 mansi368

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Posted 12 February 2013 - 01:52 AM

The point is if the construct has EF1a promoter then it must express in hematopoeitic cells as well!

pls correct me if i am wrong.

Thanks

#5 jerryshelly1

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Posted 12 February 2013 - 07:57 AM

Yes it is a human promoter, which should be constitutively expressed. I don't understand your confusion.

#6 mansi368

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Posted 12 February 2013 - 08:52 PM

The problem is i transfected the clone into hematopietic cells (PBMCs) and also in a monocytic cell line but i didnt get an overexpression of the intended gene.

#7 Ambinlab

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Posted 25 February 2013 - 09:19 PM

Hi,
sorry. I have a small question which is kind of related to this topic. Is EF1alpha promoter stronger than hUBC? If I have a lentiviral vector with GFP under UBC promoter and infect cells with very low MOI, would it lead to a good green fluorescence, which can be detected under the microscope?
Thanks

#8 almost a doctor

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Posted 26 February 2013 - 01:26 AM

The problem is i transfected the clone into hematopietic cells (PBMCs) and also in a monocytic cell line but i didnt get an overexpression of the intended gene.


Are you certain your transfection is working? Do you have positive controls, or some way to confirm that is an expression fail and not a transfection fail?

More details on what you are actually doing will help us help you.

#9 GNANA

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Posted 26 February 2013 - 03:46 AM

just to add up, did you checked the expression other than fluorescence microscope? is it empty GFP or having gene or interest tagged with GFP?. if its empty GFP you should able to visualize under fluorescence microscope directly on grown culture dish with medium, on the contrary if its tagged with other proteins, sometimes the signal wnt be in reach under flurescence microscope in the presence of medium considering the localization of your GOI, if this is the case better grow them into chamber slides and check the expression under confocal microscope rather than checking them directly in culture dish with medium under fluorescence microscope.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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